Molecular Biology

Molecular Genetics

2007-12-31T23:59:00.000-08:00

This site deals with Molecular Genetics, particularly in relation to genetic mechanisms of biological evolution. The site is searchable using the "Search this blog" box at top left, though Google is slow to update the search. Blue terms link to explanatory items so that you may navigate through items providing more detail, and the site is hyperlinked to the Companion Sites listed in the sidebar [indices]. The Molecular Genetics Overview or the SITE MAP lead into the items. Use the "back" function to return to each departure item.

Molecular Genetics Overview

2007-12-31T23:58:00.000-08:00

Molecular genetics is the study of molecules and mechanisms involved in genetic inheritance. Archival information molecules are long polymers of deoxyribonucleic acid (DNA) comprising bases in specific sequence. The bases adenine (A), thymine (T), cytosine (C), and guanine (G), function as codon triplets – sequences of three bases that code for specific amino acids or for translation initiation (start codons) or termination (stop codons). Uracil is substituted for thymine in RNA.

The segments of DNA that contain protein-coding instructions are called genes, and these gene sequences comprise a portion of the total genome of a cell. The genome includes both the genes (coding-sequences, domains) and the non-coding sequences – both exons, which include open reading frames, and introns.

Because the 64 possible combinations of GATC code for only the 20 amino acids commonly found in proteins, the code is 'degenerate' (redundant) with more than one triplet combination coding for each amino acid. (This code reduncancy provides hereditary stability by reducing mutation mistakes.) The double helix of DNA comprises paired nucleotide strands with bases hydrogen bonded to complementary bases in the adjacent chain. Adenine pairs with thymine or uracil (A-TU), and cytosine pairs with guanine (CG).

During cellular reproduction, strands of archival DNA are copied or replicated. Transcription is the first step in gene expression – DNA instructions are converted into mRNA codons, rRNAs, miRNAs, and tRNAs. Coding instructions of nucleotide sequences in archival DNA, which have been transcribed and processed into mRNAs are translated into polypeptides and proteins at cytoplasmic ribosomes. Translation is the ultimate step in gene expression, in which archival genetic instructions are converted into specified sequences of amino acids in peptides, polypeptides, and proteins.

In prokaryotic cells – without a nuclear membrane – translation into polypeptides and proteins may begin prior to termination of transcription. The molecular genetics of eukaryotic cells is more complicated than that of prokaryotes. Various molecules of ribonucleic acid (RNA) participate in the transcription of the DNA code into processed mRNA in a series of RNA processing stages including capping, polyadenylation, and pre-mRNA splicing.

Following pre-mRNA processing, RNAs undergo extranuclear transfer. Mature RNAs may undergo post-transcriptional modulation (via miRNAs) before translation of the archival DNA instructions into specific sequences of amino acids in the polypeptides and proteins that participate in cellular function and structure. Transfer RNAs (tRNA) deliver specific amino acids to the cytoplasmic ribosomes along the rough endoplasmic reticulum. Ribosomal RNAs participate in assembly of polypeptides and proteins at ribosomes. Here RNAs serve as ribozymes – non-protein enzymes.

A number of processes are involved in control of cellular function through the maintenance of accuracy of genetic inheritance – damage to DNA is repaired, and faulty RNA is destroyed.

DNA damage may result from replication errors, incorporation of mismatched nucleotides (substitution errors – transitions and transversions), damage by oxygen radicals, hydroxyl radicals, ionizing or ultraviolet radiation, toxins, alkylating agents, and chemotherapy agents. A number of vital mechanisms repair DNA damage to bases (including C to T, C to U, and T U mismatch) and to strands, including double strand breaks. All organisms, prokaryotic and eukaryotic, utilize at least three enzymatic excision-repair mechanisms for damaged bases: base excision repair, mismatch repair, and nucleotide excision repair.

Given the importance of mRNA as an information-carrying molecule, faulty pre-mRNAs and mRNAs must be eliminated – they are destroyed by nonsense-mediated decay or nonstop decay:
1. A pre-mRNA made from a mutant gene usually has an exon junction complex (EJC) in the wrong position. This error activates nonsense-mediated decay (NMD) and destroys the pre-mRNA before it can be used to make flawed proteins. There are at least two kinds of NMD: one requires the protein UPF2 and the other does not.
2. Nonstop decay is mRNA turnover mechanism that has none of the properties of normal mRNA turnover or of NMD. A multi-enzyme complex called the exosome is important for nonstop decay. The exosome is the site for binding of a specific adapter protein called Ski7p. Nonstop decay shares none of the enzymes required for nonsense-mediated decay.

Just as cells repair DNA, they must also maintain the proteome by managing damaged proteins. Heat stress denaturates proteins, causing weakening of polar bonds and exposure of hydrophobic groups. The cellular stress response (heat-shock response) protects organisms from damage resulting from environmental stressors such as heat, UV light, trace metals, and xenobiotics. Stress genes are activated to rapidly synthesize stress proteins, which are highly conserved in biological evolution and play similar roles in organisms from bacteria to humans. Normally, several constitutive stress proteins are present at low levels to function as molecular chaperones, so as to facilitate folding, assembly, and distribution of newly synthesized proteins. For the environmentally stressed cell, stress proteins protect and repair vulnerable protein targets, and play a role in the lysosomal and ubiquitin protein degradation pathways (for removal of unsalvageable proteins). Thus, the cellular stress response performs orchestrated induction of key proteins necessary for cellular protein repair and degradation systems.

alternative splicing

2007-12-31T23:19:00.000-08:00

Alternative splicing is a carefully regulated, variable adaptation of the constitutive RNA modification process of pre-mRNA splicing. Alternative splicing is a form of epigenetic mechanism that enables a single gene to give rise to multiple, differentially spliced versions of a protein, increasing complexity without a change in the genome. ... MORE

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transcription

2007-12-31T23:11:00.000-08:00

In transcription, an RNA polymerase enzyme (RNAp, or pol III in eukaryotes) directs generation of a complementary strand of mRNA from DNA. The mechanism of alternative splicing enables to production of different mature mRNA molecules, depending on what sequences are treated as introns and what remain as exons.

Transcription involves 3 phases: initiation, elongation, and termination.
1. RNA polymerase II initiates transcription at the first nucleotide of the first exon of a gene.
2. The 5 end of the nascent RNA is capped with 7-methylguanylate (capping).
3. Transcription by RNA polymerase II terminates at any one of multiple termination sites downstream from the poly(A) site, which is located at the 3 end of the final exon.
4. After the primary transcript is cleaved at the poly(A) site,
5. A string of adenine (A) residues is added (polyadenylation). The poly(A) tail contains ≈250 A residues in mammals, ≈150 in insects, and ≈100 in yeasts.

In more detail: RNA polymerase binds to the promoter region of one strand of DNA (5’ end), and the DNA double helix is un-zipped into single strands. First, RNA polymerase requires a number of general transcription factors (called TFIIA, TFIIB, etc.). The promoter contains a DNA sequence called the TATA box, which is located 25 nucleotides away from the site of initiation of transcription. The TATA box is recognized and bound by transcription factor TFIID, which then enables the adjacent binding of TFIIB. The rest of the general transcription factors plus the RNA polymerase assemble at the promoter. diagram - initiation of transcription.

The RNAp enzyme moves toward the 3’ end, connecting complementary bases into an elongating chain of RNA nucleotides. At termination, the transcribed mRNA molecule is released from the DNA strand. In prokaryotic cells – without a nuclear membrane – translation may begin prior to termination. In eukaryotic cells – with a nuclear membrane – the processed mRNA moves through the nuclear pores into the cytoplasm, where ribosomes on the rough endoplasmic reticulum translate the mRNA code into a peptide or a protein. Epigenetic, alternative splicing mechanisms can edit the mRNA prior to its translation into protein.

Capping of the 5’ end on the pre-mRNA with 7-methylguanylate occurs soon after initiation of transcription, and the 5 cap is retained in mature mRNAs.

Cleavage, pre-mRNA splicing, and polyadenylation usually follow termination of transcription of short primary transcripts with few introns. However, introns often are spliced out of the nascent RNA before transcription of the gene is complete for large genes with multiple introns.

It was believed that most genes in higher eukaryotes are regulated by controlling their transcription. However, it is increasingly recognized that epigenetic mechanisms (such as alternative splicing) are important in generating many proteins from a single gene, accounting for the Human Genome Project’s discovery that a mere 30,000 genes code for about 100,000 proteins.

animation - start of transcription : animation - life cycle of an mRNA : animation ~ alternative splicing More at NCBI Molecular Cell Biology - Transcription Initiation Complex : SUMMARY transcription initiation (NCBI MCB) : Processes of Transcription : Wikipedia : Central Dogma :

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base excision repair

2007-12-29T23:56:00.000-08:00

Base excision repair (BER) describes one form of excision repair in which damaged bases or incorrect bases are excised and replaced through specific enzymes that differ between species. However, the biochemical processes involved in BER are equivalent across species, so bacterial DNA repair functions can operate in eukaryotic cells, and vice versa. Damage is typically the result of deamination, alkylation, hydroxylation, or attack by an oxygen radical, while the incorrect base can be uracil substituted for thymine. Oxidative DNA lesions induced by oxygen free radicals such as superoxide and hydroxyl radicals appear to be repaired predominantly by base excision repair mechanisms. Further, BER is the major DNA repair system involved in removal of various oxidative DNA lesions induced by ionizing radiation - these include abasic sites and modified DNA base and sugar residues.

Left - diagram of base excision repair - click to enlarge image.
NTPs = ribonucleoside triphosphates
dNMP = deoxyribonucleoside monophosphate

First, the altered base is excised by a specific DNA glycosylase, which breaks the beta N-glycosidic bond and creates an AP, or abasic site. This site is identical to that generated by spontaneous depyrimidination or depurination. Six DNA glycosylases have been identified in humans – each excises an overlapping subset of either spontaneously formed (such as hypoxanthine), oxidized (such as 8-oxo-7,8-dihydroguanine), alkylated (such as 3-methyladenine), or mismatched (for example, T:G) bases.

Next, the terminal sugar-phosphate is removed by an AP endonuclease (Ape1), leaving a 3’-OH terminal and an abnormal 5'-abasic terminus. The resulting gap is refilled by the 5’-deoxyribose-phosphodiesterase action of a DNA polymerase I (DNA polymerase beta in mammals), then the strands are re-ligated by DNA Ligase I or a complex of XRCC1 and LigIII.

An alternative BER pathway corrects errors involving more than one nucleotide. The Fen1 protein excises the long-patch structure that is produced by DNA polymerase strand displacement. This "long-patch" repair process is divided into two subpathways: a PCNA-stimulated, Pol-beta-directed pathway and a PCNA-dependent, Pol-delta/epsilon -directed pathway.

link to table - human DNA repair genes : diagram>BER

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cis versus trans-acting factors

2007-12-28T23:36:00.000-08:00

Most often, signal elements act only on the intramolecular nucleotide sequence to which they are attached, and they are said to act "in cis". Intron removal in eukaryotes involves cis-splicing. Interaction with signal factors -- usually protein molecules -- turns signal elements on or off.When protein factors are free to diffuse within the cell they can act on target elements that may not be derived from the same genome segment. Protein factors capable of acting upon other intermolecular genome segments are called "trans-acting factors".

One form of trans-splicing is the 'spliced leader' type, which is primarily found in protozoans (e.g. trypanosomes) and in lower invertebrates such as nematodes. This results in the addition of a capped, noncoding, spliced leader sequence to the 5' end of mRNAs.

Another form of trans-splicing is the 'discontinuous group II intron' type that occurs in plant/algal chloroplasts and plant mitochondria. This results in the joining of two independently transcribed coding sequences. Both spliced-leader and discontinuous group II intron trans-splicing are mechanistically similar to conventional nuclear pre-mRNA cis-splicing. Trans-splicing also occurs in mammalian cells, just as cis-splicing occurs in trypanosomes. It has been suggested that both trans- and cis-splicing are ancient acquisitions of the eukaryotic cell. (Abstract)

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capping

2007-12-28T23:27:00.000-08:00

Capping is a form of RNA processing in which the 5’ end of the nascent pre-mRNA is capped with a 7-methyl guanosine nucleotide, 7-methylguanylate. Capping occurs shortly after initiation of transcription.

The 5' cap is retained in mature mRNAs. Capping is required to protect the RNA transcript from degradation. It plays an important role in mRNA transport to the cytoplasm and in the initiation of protein synthesis (translation) .

life cycle of an mRNA ~ click on Quicktime Q NCBI Molecular Cell Biology Post-transcriptional Processing of RNAs

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codon

2007-12-28T23:22:00.000-08:00

Triplets of DNA nucleotides arranged in sequence along DNA are transcribed into the codons of mRNA strands.

In the centre of the DNA double helix each nucleobase (base) is hydrogen bonded to its complementary base – because of steric (size) constraints, purines bases pair with pyrimidine bases. A with T, and C with G.

RNA molecules are created by transcription from the archival DNA template. Like replication, transcription proceeds 3' to 5' on the template strand and 5' to 3' on the RNA strand. Proteins are synthesized from the amino acid terminus to the carboxyl terminus by formation of peptide bonds between amino acids delivered by tRNA anticodons.

Because the nucleotide triplet sequences would produce different amino acids if ‘reading frames’ commenced at the wrong nucleotide, codons in genes are read in reading frames that commence at the start codon – usually the first AUG in the RNA sequence (ATG triplet for DNA). AUG also codes for the amino acid methionine.

The RNA code is at left (click to enlarge). In accord with its origin early in evolution, the genetic code is almost universal – with only a few organisms employing variants.

Each tri-nucleotide in the DNA sequence and transcribed mRNA codon signals for the insertion of one amino acid into a polymerizing peptide chain. The sequence of codons in genes determines the sequence of amino acids inserted into peptide, polypeptide and protein chains (primary structure).

Take an interactive peek at a sequence of DNA around the gene cyclooxygenase 2: PBS Cracking the Code of Life : Find on/off : Find start : Find stop : Find exons : Find introns : Find transposable elements : Find conserved DNA : Find single nucleotide polymorphisms : Find gene and scroll down:

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chromosome

2007-12-28T16:08:00.000-08:00

Genetic archival material is condensed into chromosomes (Left - click to enlarge image).

1. one of two sister chromatids
2. centromere (kinetocore)
p. short 'p' arm of chromatid
q. long 'q' arm of chromatid

A chromosome contains a long strand of deoxyribonucleic acid containing genes, regulatory sequences, and non-coding sequences of nucleotides, in association with proteins. The full chromosomal complement of a cell comprises the genome, which is the complete hereditary information of an organism contained within macromolecules of archival DNA .

The multiple nuclear chromosomes of eukaryotes comprise long helical strands of DNA wrapped around structural proteins called histones – this composite material is termed chromatin (diagram). Each eukaryotic chromosome comprises one or two (sister) chromatids (1), each with a kinetochore (2) for attachment to a microtubule of the spindle apparatus during cell division. Chromatids have a long (q) and a short (p) arm attached to the centromere (2). Sister chromatids attach to each other, or to the spindle apparatus, by means of special proteins and DNA base sequences in the kinetochore region.

Chromosomes (Gk. 'colored bodies') are most visible during metaphase (tem), and least condensed (dispersed) when participating in expression (tem, tem2) such as occurs in cells with large undifferentiated nuclei (colored tem cancer cell, fluorescence microscopy of cancer, stem cells, immature cell with oncogene (black dots) ).

Prokaryotes mostly possess one or two* chromosomes termed nucleoids (tem). Prokaryotes lack a membrane enclosed nucleus and their DNA is usually contained in circular structures located within the cytosol, but may be organized as linear strands that are typically attached to the plasma membrane. Plasmids are small circular, extrachromosomal genetic elements that can be transmitted from one bacterium to another through pili during conjugation. (more)

Chromatin (DNA plus histone protein) exists in two basic forms:
1. Euchromatin, from which DNA is being actively transcribed (expressed) into RNA for ultimate translation into polypeptide and protein molecules.
2. Heterochromatin, which consists of either:
a. Facultative heterochromatin, which is sometimes expressed.
b. Constitutive heterochromatin, which is located around the centromere and usually contains repetitive sequences, and which is never expressed.

*for example, Vibrio cholerae and Deinococcus radiodurans

DNA polymerases

2007-12-27T23:26:00.000-08:00

MOLECULAR BIOLOGY: ON DNA POLYMERASES: "Evolution has produced a number of different types of DNA polymerase, but they all have a similar overall three-dimensional shape that has been likened to a right hand, with palm, finger and thumb domains [1]. Polymerases of the A and B families, such as polymerase d, replicate the bulk of genomic DNA during the cell cycle and have been streamlined for processivity and accuracy. These enzymes fit the DNA substrate tightly into their active site, where the replicating base pair is enclosed by the finger domain [2-4]. The mobility of the finger domain underlies a so-called 'induced-fit' mechanism for checking the fidelity of replication: only when an incoming nucleotide forms a perfect Watson-Crick pair with the template base can the fingers close and induce an active conformation of the polymerase. If, nevertheless, an erroneous nucleotide happens to be incorporated, the polymerase responds with conformational distortions of its active center. These induce replication pausing and translocation of the primer terminus towards the intrinsic 'proofreading' exonuclease activity, which removes the mispairing base so that synthesis can resume." O. Fleck and P. Schär (Current Biology 2004 14:R389)

Carver, T.E.Jr. , Hochstrasser, R.A., and Millar, D.P. (1994). Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I. Proc. Natl. Acad. Sci. USA 91, 10670-10674

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DNA

2007-12-27T23:25:00.000-08:00

DNA is deoxyribonucleic acid, the template for genetic instructions. DNA undergoes replication into complementary strands of DNA for reproduction, and transcription into RNA before translation into proteins. The nucleotide bases, adenine, thymine, cytosine, and guanine are coupled inside the double helix, while the ribose backbone curves around the edge. (click images to enlarge)

Right - nucleobases form complementary pairs through hydrogen bonding. Adenine and thymine are coupled (broken lines), and cytosine couples with guanine.

grey - carbon / white - hydrogen / blue - nitrogen / red – oxygen / green - phosphorus

More at: http://www.dnaftb.org/dnaftb/ : http://www.dnalc.org/home.html : http://www.pbs.org/wgbh/aso/tryit/dna/

More detail pending:
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DNA repair

2007-12-27T23:24:00.000-08:00

dNTP: "In eukaryotes, DNA damage elicits a multifaceted response that includes cell cycle arrest, transcriptional activation of DNA repair genes, and, in multicellular organisms, apoptosis."

Damage to DNA can be caused by mutations such as replication errors, incorporation of mismatched nucleotides (substitution errors -- transitions and transversions) DNA damage can also result from unintentional and intentional environmental stimuli such as oxygen radicals, hydroxyl radicals, ionizing or ultraviolet radiation, toxins, alkylating agents, and chemotherapy agents, particularly anti-cancer drugs.

Damaged bases can be corrected by simple in situ chemical reversal of the defect, but excision-repair processes predominate. These important DNA repair mechanisms take advantage of the fact that DNA is double-stranded and that complementary sequences should match on both strands. So, if damage is confined to one strand, the damage can be accurately repaired by excision and replacement with DNA synthesized with the undamaged complementary strand acting as template. All organisms, prokaryotic and eukaryotic, utilize at least three enzymatic excision-repair mechanisms: base excision repair, mismatch repair, and nucleotide excision repair.MOLECULAR BIOLOGY: ON DNA-REPAIR ENZYMES

The DNA-repair enzymes have the capability of searching through vast tracts of DNA to uncover subtle structural anomalies. The human repair enzyme 8-oxoguanine glycosylase (hOGG1) efficiently removes 8-oxoguanine (oxoG), a damaged guanine (G) base containing an extra oxygen atom, while it ignores undamaged bases. The structure of hOGG1 bound to undamaged DNA, reveals a unique strategy that permits faithful removal of damaged bases which do 'fit' into the oxoG slot at the enzyme's active site, while normal G bases do not.

Anders Jahres medisinske priser: modified: "To date, at least six distinct pathways of DNA repair have been discovered, comprising nucleotide excision repair , base excision repair , mismatch repair , repair by recombination (homologous and nonhomologous end rejoining), damage tolerance pathways (polymerase bypass) and different damage reversal mechanisms, involving close to 200 genes in human cells. "

PLoS Biology: Three New Phases of Repairing DNA Damage in E. coli: "E. coli SOS response has been used to study DNA repair for decades, and a great deal is known about how the more than 30 genes involved in the response function. Two proteins figure prominently in this response. The LexA protein acts as a repressor and inhibits the expression of SOS genes under normal conditions; in the event of DNA damage, the protein RecA inactivates the LexA repressor by enhancing its autocleavage into two fragments, which initiates the SOS response. "

Discovery Of Why Some DNA Repair Fails: Significant For Huntington's Disease And Colon Cancer: "Dr. McMurray's group studied a specific mismatch repair protein Msh2-Msh3 and found a paradox: Instead of helping repair DNA damage, under certain conditions, Msh2-Msh3 was actually harming the cell. Msh2-Msh3 did this when it arrived at the wrong place at the wrong time and bound to a specific portion of DNA (CAG-hairpin). This accident of binding at the CAG-hairpin altered the biochemical activity of Msh2-Msh3. This change in biochemical activity, in turn, promoted DNA expansion -- rather than repair -- and changed the function of Msh2-Msh3 from friend of DNA to foe by allowing damaged DNA to go unrepaired. Without DNA repair, mutations accumulate that lead to disease."

MOLECULAR BIOLOGY: ON DNA-REPAIR ENZYMES: "hOGG1 makes extensive contacts with the orphaned cytosine base, which ensures that oxoG is removed only when in the appropriate base-pairing context. Although extensive biophysical and structural studies intimate that there are general features of damaged bases that signal their presence to repair enzymes, the steps involved in finding damaged bases in a sea of normal ones are still unclear. Most mechanisms invoke the enzyme sliding or hopping along the DNA duplex until a damaged site is detected. A particularly intriguing question is whether normal bases are also extruded from the helix during the search process."

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DNA damage by ROS

2007-12-27T23:23:00.000-08:00

Measurement of Oxidative DNA Damage and Repair in the Aging and Diseased Human Brain Using Liquid and Gas Chromatography/Mass Spectrometry with Isotope Dilution: "There is accumulating evidence that reactive oxygen species (ROS) play an important role in aging and neurodegenerative disease. Genomic DNA appears to be a particularly important target for ROS, since human patients and knockout mice lacking the capacity to repair certain types of [sic] oxidative DNA damage experience neurodegeneration and show evidence of premature aging."

More detail pending:
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targetted genetic repair

2007-12-27T23:21:00.000-08:00

Targeted genetic repair: an emerging approach to genetic therapy -- Sullenger 112 (3): 310 -- Journal of Clinical Investigation: "Targeted gene repair is a powerful yet controversial technique developed to direct base changes in chromosomal genes, while RNA repair is an emerging strategy to alter the coding content of messenger RNAs.Genetic repair strategies may have significant therapeutic and safety advantages over traditional gene therapy approaches for the treatment of many genetic disorders. Firstly, because the mutant genetic instructions are directly repaired, the corrected RNAs and/or DNAs will be maintained in their native sequence context and be regulated by their endogenous regulatory machinery. Secondly, in the instance where the mutant gene encodes a deleterious or dominant-negative mutant protein, repair of the mutant should simultaneously engender the regulated production of the wild-type protein while eliminating or reducing expression of the deleterious gene product. Finally, genetic repair strategies attempt to repair defective instructions in a site-specific manner. Therefore, once adequately developed, these strategies will result in less random mutagenesis of the genome and lead to fewer mutagenic side effects than do methods that randomly insert genes into the genome. "J. Clin. Invest. 112:310-311 (2003). doi:10.1172/JCI200319419. Perspective : Targeted genetic repair: an emerging approach to genetic therapy.

Related Free Full Text articles:
Hacein-Bey-Abina, S. et al.2003. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348:193-194.[Free Full Text]
Long, M.B., Jones, J.P. III, Sullenger, B.A., and Byun, J. 2003. Ribozyme-mediated revision of RNA and DNA. J. Clin. Invest. 112:312-318. doi:10.1172/JCI200319386.[Free Full Text]
Lan, N. et al.1998. Ribozyme-mediated repair of sickle ß-globin mRNAs in erythrocyte precursors. Science. 280:1593-1596.[Abstract/Free Full Text]
Watanabe, T., and Sullenger, B.A. 2000. Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts. Proc. Natl. Acad. Sci. U. S. A. 97:8490-8494.[Abstract/Free Full Text]
Rogers, C.S., Vanoye, C.G., Sullenger, B.A., and George, A.L. 2002. Functional repair of a mutant chloride channel using a trans-splicing ribozyme. J. Clin. Invest. 110:1783-1789. doi:10.1172/JCI200216481.[Abstract/Free Full Text]
Broitman, S., Amosova, O., Dolinnaya, N.G., and Fresco, J.R. 1999. Repairing the sickle cell mutation. I. Specific covalent binding of a photoreactive third strand to the mutated base pair. J. Biol. Chem. 274:21763-21768.[Abstract/Free Full Text]
Vasquez, K.M., Narayanan, L., and Glazer, P.M. 2000. Specific mutations induced by triplex-forming oligonucleotides in mice. Science. 290:530-533.[Abstract/Free Full Text]

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double strand breaks

2007-12-27T23:13:00.000-08:00

MRC Radiation and Genome Stability Unit: "DNA double-strand breaks (DSBs) are potentially lethal, recombinogenic lesions which can result from exposure to DNA damaging agents such as ionising radiation or endogenous events such as collapsed replication forks. Failure to correctly repair a DSB can result in cell death or tumorigenesis. Indeed many cancer genes have been found to function in DSB metabolism. Eukaryotic cells mount a coordinated response to DSBs which includes cell cycle arrest, DNA repair and transcriptional stress responses. These responses are controlled by the DNA integrity checkpoint, DNA repair and stress-activated MAP kinase pathways, respectively. "

More detail pending:
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epigenetics

2007-12-26T23:15:00.001-08:00

Defining epigenetic states through chromatin and RNA - Nature Genetics: "The term 'epigenetics' is used to describe heritable changes in genome function that occur without a change in DNA sequence. As such, epigenetics lies at the heart of the cellular memory crucial for development and provides an important avenue for sustained response to environmental stimuli."

More detail pending:
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enhancer

2007-12-26T23:15:00.000-08:00

An enhancer is a short DNA sequence that increases the level of expression of another gene, that is, the enhancer up-regulates transcription of genes within the regulated gene-cluster. Specific trans-acting, transcription factors bind to the enhancer to bring about the increase in transcription rate -- recruiting the initiation complex proteins, or stabilizing the initiation complex.Because of looping of the DNA strands, there may be a separation of several thousand base pairs between the enhancer and initiator gene (start site). However, the enhancer and its regulated gene are located on the same chromosome.The enhancer segment may be situated upstream or downstream of the enhanced gene, and its orientation is not fixed – that is, the enhancer’s sequence may be reversed without altering its function. Enhancer segments may be excised and repositioned without interrupting their regulatory function. Enhancers may occur within introns. Enhancers cause the opposite effect to that of silencers which repress transcription.

Regulatory Genes & Enhancers
ScienceMatters @ Berkeley. Fly Guy: "Regulatory DNA, Levine explains, controls how and where a gene is expressed in a cell. Of the three types of regulatory DNA--enhancer, silencer, and insulator--'enhancers are king, activating gene expression in specific cell types for specific tissues,' he says. Scientists conservatively estimate that while the human genome has less than 30,000 genes, it may contain 100,000 enhancers at the minimum. So far, just 50 or so have been identified."

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exon

2007-12-26T23:04:00.000-08:00

Exons are sequences of the primary RNA transcript (or of the DNA that encodes them) that exit the nucleus as part of a messenger RNA molecule. In the primary transcript, neighbouring exons are separated by introns, which must be removed during pre-mRNA splicing .”

In other words, exons are those sections of DNA within a gene that are not spliced out from the transcribed precursor mRNA (pre-mRNA) and that are retained in the final messenger RNA (mRNA) molecule. For many genes, each exon contains part of the open reading frame (ORF) that codes for a specific portion of the complete protein. However, the term exon is often misused to refer only to coding sequences for the final protein. This is inaccurate since many noncoding exons are known in human genes (Entrez).

The term "exon" was coined by Walter Gilbert in 1978. Gilbert shared the 1980 Nobel Prize in Chemistry with Paul Berg and Frederick Sanger.

The Exon-Intron Database

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FAS gene

2007-12-25T23:05:00.000-08:00

The FAS gene encodes one of several proteins important to apoptosis, a process referrred to as programmed cell suicide. FAS is now officially designated tumor necrosis factor receptor superfamily member 6 (TNFRSF6). Other synonyms for the receptor are FASLG receptor, ALPS1A, apoptosis-mediating surface antigen, Apo-1 antigen, APT1, and CD95 antigen, FAS1, FASTM.

: ALPS : cancer : caspase 8, caspase 10 : death-inducing complex : DISC : FADD : Fas-associated death domain protein : MAPK3/ERK1 : MAPK8/JNK : NF-κβ : peripheral tolerance : T cells, T-cells :

The adapter molecule FADD recruits caspase-8 to the activated receptor, and the resulting death-inducing signaling complex (DISC) performs caspase-8 proteolytic activation, which initiates the subsequent cascade of caspases (aspartate-specific cysteine proteases).

The protein encoded by the FAS gene is a member of the TNF-receptor superfamily. The TNF-receptors contain a death domain and play a central role in the physiological regulation of programmed cell death (apoptosis). The interaction of TNF-receptor and ligand causes the formation of a death-inducing signaling complex that includes the adapter molecule, Fas-associated death domain protein (FADD), caspase 8, and caspase 10. The autoproteolytic processing of the caspases in the FADD complex triggers a downstream caspase cascade, leading to apoptosis.

TNF-R activates NF-κβ, MAPK3/ERK1, and MAPK8/JNK. It is involved in transducing the proliferating signals in normal diploid fibroblast and T cells. At least eight alternatively spliced transcript variants, encoding seven distinct isoforms, have been described and the isoforms lacking the transmembrane domain may negatively regulate the apoptosis mediated by the full length isoform.

FAS-mediated apoptosis may have a role in the induction of peripheral tolerance, in the antigen-stimulated suicide of mature T-cells, or both. TNF-Rs are implicated in the pathogenesis of immune system diseases and various cancers (T-cell ALL, multiple myeloma, lung cancer, malignant melanoma, bladder cancer, esophageal cancer, Ewing's sarcoma, etc).

FAS mutations are found in 83% of cases of ALPS (the autoimmune lymphoproliferative syndrome), an autoimmune condition in which lymphocytes fail to undergo apoptosis on schedule. The FAS gene is located on chromosome 10q24.1, and is member 6 of the tumor necrosis factor receptor superfamily.

: ALPS סּ apoptosis ф autoimmunity : cancer : caspase 8, caspase 10 סּ caspases : death-inducing complex סּ death receptor : DISC : FADD : Fas-associated death domain protein ¤ malignant transformation : MAPK3/ERK1 : MAPK8/JNK : NF-κβ : peripheral tolerance סּ receptor proteins : T cells, T-cells ф T cells :

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genes

2007-12-24T23:47:00.000-08:00

The genes are those portions of the genome that code for the production of proteins. Typically protein coding segments are found in the open reading frames of introns, though alternative splicing and other epigenetic modifications account for much of the complexity of the genome.

More detail pending:
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genome

2007-12-24T23:46:00.000-08:00

The term genome refers to the complete hereditary information of an organism (archival DNA or RNA for some viruses). The genome includes both the genes (coding-sequences, domains) and the non-coding sequences – both exons, which include open reading frames, and introns. Similarly, the term proteome refers to an organism’s collection of proteins.

The genome possesses:
1. Exonal segments of DNA whose sequence encodes the pre-mRNA, and ultimately polypeptide and protein sequences.
2. Intronal segments that are excised by pre-mRNA splicing before transport of mature mRNA through nuclear pores to the cytoplasm where ribosomal translation into ribosomal polypeptides and proteins occurs.
3. A start site for transcription, the initiator gene.
4. Promoters, both a basal or core promoter located within about 40 bp of the start site, and an upstream promoter, which may extend over as many as 200 bp farther upstream.
5. Enhancers.
6. Insulators.
7. Silencers.

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gene regulation

2007-12-24T23:45:00.000-08:00

Gene regulation mechanisms in eukaryotes, which possess a nuclear membrane, differ from those in prokaryotes. Because prokaryotes lack a nuclear membrane, simultaneous translation of a gene may commence before transcription is complete.

In eukaryotes mechanisms for control of gene expression:
a. Most commonly affect the rate of transcription.
b. Some alter the rate of RNA processing within the nucleus.
c. Some affect the stability and degradation of RNA molecules (nonsense-mediated decay, nonstop decay).
d. Some control the efficiency of ribosomal translation into ribosomal polypeptides and proteins.
e. Some allow for alternative splicing, which generates different proteins from the same archival DNA template.
f. Epigenetic mechanisms modify mRNAs.

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helicases

2007-12-23T23:47:00.000-08:00

"Since the discovery of the 'DNA unwinding enzyme,' it has become clear that helicases participate in virtually all cellular processes involving nucleic acids. Helicases are found in all three kingdoms of life and are extremely numerous: 1-2 percent of eukaryotic genes are helicases. Several severe human genetic diseases have been linked to mutations in helicases. . . The most fundamental activity for all helicases is translocation, the ability to move along nucleic acids. Translocation is powered by ATP hydrolysis; hence helicases are motor proteins. Many helicases function as a part of large macromolecular complexes. An example is chromatin remodeling, which regulates gene expression by controlling the DNA access in chromatin. Helicases are the central ATP-powered engines that drive the translocation of the chromatin-remodeling complexes along the DNA. . . Ha's lab measures FRET (fluorescence resonance energy transfer) between various sites on the protein and on the DNA to build dynamic structural models of the protein-DNA complex during translocation."

HHMI News: DNA Enzyme Shows Unexpected Acrobatic Flair: "Helicase Protein: A helicase protein moving rapidly on a highly flexible single-stranded DNA track. Repetitive movement on the DNA may keep it clear of potentially toxic proteins. Watch Animation at Helicase 8KB Flash Animation(requires Flash Player) "

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heterochromatin

2007-12-23T23:45:00.000-08:00

MOLECULAR BIOLOGY: CHROMATIN DNA PACKAGING AND GENE SILENCING
Cytological studies have demonstrated that much of the repetitious DNA is packaged in a condensed form referred to as heterochromatin. This packaging in the nucleosome limits transcription by rendering DNA segments inaccessible.

The packaged mode of heterochromatin is epigenetically inherited, in that the packaging state is typically maintained after replication and mitosis, independent of the underlying DNA sequence. This property implies a that a biochemical mark exists together with cellular machinery that can recognize and maintain the mark locally.

The heterochromatin of all eukaryotes is characterized by histone hypoacetylation, and by methylation of histone H3 on lysine 9 in higher eukaryotes. Some single-celled eukaryotes such as Saccharomyces lack methylation of H3. Heterochromatin Protein 1 (HP1) binds H3 methylated at lysine 9 (H3-mK9). HP1 is a highly conserved protein that directly associated with pericentric heterochromatin.

A Unified Mode of Epigenetic Gene Silencing RNA Meets Polycomb Group Proteins A Unified Mode of Epigenetic Gene Silencing: Article Abstract: "Recently, an essential role for RNA in the epigenetic silencing of genes packaged within heterochromatin in animals has been recognized. The RNA appears to be involved in targeting chromatin remodeling activity to specific loci and in later maintaining the repressed state of the gene. Epigenetic silencing of Hox cluster genes by the Polycomb group proteins also involves the formation of a stably inherited repressive chromatin structure. Recent studies of the C. elegans PcG gene sop-2 revealed an evolutionarily conserved property of PcG proteins in the binding of RNA, suggesting an important role for RNA in PcG-mediated Hox gene repression."

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insulator

2007-12-23T23:23:00.000-08:00

An insulator is a section of DNA (40 bp or more) that is located between the enhancer(s) and the promoter, or between the silencer(s) and the promoter of adjacent genes or clusters of adjacent genes.

More detail pending:
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intron

2007-12-23T23:22:00.000-08:00

Because the exon segments of archival DNA are transcribed into messenger RNAs and translated into proteins, the earlier view of genes held that introns were solely ‘junk DNA’ or DNA 'deserts' because they do not contain ORFs to code for proteins. However, it has recently been recognized that some exons code for micro RNAs and represent a source of epigenetic coding.

Some segments that were formerly designated introns contain information for protein, and others code for RNA products and are thus not "junk" DNA. Other introns posses translatable nucleotide sequences that, in the absence of splicing, can generate production of novel peptides (maturases) fused to the peptide encoded by the N-terminal exons. In fungi, these peptide maturases, appear to function in intron removal. Their encoding in introns results in homeostatic regulation of their production. Maturase genes are interspersed within other genes.“

Group II introns are a novel class of RNAs (ribozymes) best known for their self-splicing reaction. Under certain in vitro conditions, the introns can excise themselves from precursor mRNAs and ligate together their flanking exons, without the aid of protein. The splicing mechanism is essentially identical to splicing of nuclear pre-mRNA introns (pre-mRNA splicing), and this similarity has led to the widespread belief that group II introns were the ancestors of spliceosomal introns, which make up 25% of the human genome.

Some group II introns have a second remarkable property: they encode reverse transcriptase (RT) ORFs and are active mobile elements. Such mobile group II introns can insert into defined sites at high efficiencies (called retrohoming), or can invade unrelated sites at low frequencies (retrotransposition).”

Ribozyme-mediated revision of RNA and DNA -- Long et al. 112 (3): 312 -- Journal of Clinical Investigation: "Group I introns are ribozymes that carry out two transesterification reactions in order to excise themselves from a precursor transcript. "

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atheism

2007-12-20T16:32:00.001-08:00

A-Deistic
Adeistic
Black Out
cosmic
Eine Kleine Nattermusing
eMusings
eVolition
Galaria
Godborygmi
Godspell Follies
Gray Matters
MetaThoughts
Mimble Wimble
Naturalism
BLogodaedaly
palimpsest
Salient
Science of Evolution
Sechuam
Sintheist
Tabula Flexuosa
The Scarlet A
Weltschauung

mismatch repair

2007-12-18T23:36:00.000-08:00

Most mismatches are caused by replication errors. However, mismatches can also be produced by other mechanisms, such as deamination of 5-methyl cytosine to generate T improperly paired to G. Where the appropriate DNA-N-glycosylase is available, mismatches can also be repaired by base excision repair.

Mismatch repair has been studied most extensively in E. coli, where the proteins MutS, MutL, and MutH initiate the repair process. Newly synthesized strands are not immediately methylated in E.coli. First, MutS recognizes and binds to true mismatches and insertions/deletions of up to 4 nucleotides. Next, MutL binds to and stabilizes this complex of MutS/mismatched strand. The MutS-MutL complex then activates MutH, which locates a nearby methyl group and creates a nick in the newly synthesized strand opposite the methyl group. Excision is accomplished in E.coli by cooperation between the UvrD (Helicase II) protein, which unwinds from the nick toward the mismatch, and a single-strand specific exonuclease of appropriate polarity. Finally, resynthesis by Polymerase III and ligation by a DNA ligase repair the sequence and re-ligate the strands.

Unlike the un-methylated new strands in E.coli, strand-specificity in eukaryotes may be signalled by single-strand nicks. In nascent eukaryotic DNA strands, single-strand breaks occur between Okazaki fragments in the lagging strand and at the 3' end of the leading strand.

Eukaryotes lack homologues of MutH and uvrD, but do possess numerous homologues of MutS and MutL (MSHs 1-6, MLHs 1-3 and PMS 1 or 2). In E.coli, MutS and MutL function as monomers. The homologous eukaryotic proteins function as heterodimers. Human cells also possess two heterodimers of MutS homologues – MutSalpha (MSH2/MSH6), which recognizes single base mismatches and small loops, and MutSbeta (MSH2/MSH3), which recognizes small loops.

In eukaryotic cells, several standard replication proteins are needed for mismatch repair. The "clamp" protein, PCNA is a cofactor for most DNA polymerases and stabilizes the MutS and MutL heterodimers at mismatch sites on DNA. Three MutL homolog dimers are also known – in humans, MLH1/PMS2, MLH1/PMS1, and MLH1/MLH3. Further, just as exonucleases are thought to be important for mismatch repair in prokaryotes, at least two nucleases appear to contribute to mismatch repair in eukaryotic cells – exonuclease 1 and Flap Endonuclease (FEN-1 or DNase IV; Rad27 in S. cerevisiae). PCNA is also required during the later DNA synthesis step of mismatch repair. The DNA synthesis step also requires RPA (the eukaryotic single-stranded DNA-binding protein), Replication factor C (which loads PCNA onto DNA molecules at primer termini) and DNA polymerase delta.

Hereditary non-polyposis colon cancer (HNPCC) is a form of colon cancer frequently associated with defects in the genes encoding MSH2 (about 35% of identified gene-defect cases) and MLH1 (about 60% of identified gene-defect cases). HNPCC is characterized by early age of onset and autosomal dominant inheritance with high penetrance.

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nonsense-mediated decay

2007-12-17T23:20:00.000-08:00

Defusing Dangerous Mutations: Scientists Discover A New Way By Which Cells Control Genetic Errors: Adapted: "Nonsense-Mediated Decay (NMD), is a process by which cells destroy potentially harmful molecules. Both healthy and damaged proteins begin as instructions in genes. Cells transcribe this information and create an mRNA molecule, a template that will be used to create proteins. RNAs usually contain extra bits of code that have to be cut out before they can be used. During this cut-and-paste operation, cells attach a group of molecules called the exon junction complex (EJC) to the RNA. An RNA made from a mutant gene usually has an EJC in the wrong position, which activates NMD and destroys the RNA before it can be used to make flawed proteins. There are at least two kinds of NMD: one requires UPF2 and the other does not.The presence or absence of UPF2 changes the composition of the EJC, giving it different surfaces to which other molecules attach. This affects the way that another component, called UPF1, fits onto the machine. UPF1 is directly responsible for calling up the NMD machinery. The study shows that UPF1 can be mounted on both EJC types; the final effect is the same – to efficiently destroy faulty RNAs. "

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nonstop decay

2007-12-17T23:19:00.000-08:00

Researchers Discover New Mechanism That Targets And Destroys Abnormal RNA:Adapted: "Messenger RNA molecules are the genetic templates for proteins. In constructing proteins, the mRNA template is transcribed from DNA genes and transported to the ribosomes -- the cell's protein "factories" that are large complexes of protein and RNA. Given the importance of mRNA as an information-carrying molecule, the machinery that regulates mRNA levels and destroys faulty mRNA is critical in ensuring that errors in the genetic code are not passed on to proteins.

Nonstop decay is mRNA turnover mechanism that has none of the properties of nonsense-mediated decay (NMD), or of normal mRNA turnover in the cell. Nonstop decay shares none of the enzymes required for nonsense-mediated decay. A multi-enzyme complex called the exosome is important for nonstop decay, site of binding of a specific adapter protein called Ski7p occurs."

One percent of genes in both humans and yeast produce mRNAs containing specific sequences that would trigger degradation of the RNA by nonstop decay. Nonstop mRNA transcripts might be important in enabling production of shortened proteins that are needed at specific stages of development. At later stages of development, when these truncated proteins are no longer needed, their mRNA could easily be destroyed by nonstop decay."
The original news release can be found here.

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nucleic acids

2007-12-17T23:10:00.000-08:00

A nucleic acid is a large biochemical macromolecule composed of chains of nucleotides coding genetic information. The most common nucleic acids are deoxyribonucleic acid ( DNA ) and ribonucleic acid ( RNA ). Nucleic acids are found in all living cells – karyotes and eukaryotes – and in viruses. Monomeric nucleic acids are called nucleotides – each consists of a nitrogenous heterocyclic base (either a purine or a pyrimidine), a five-ring pentose sugar, and a phosphate group.

More detail pending:
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nucleosome

2007-12-17T23:09:00.000-08:00

MOLECULAR BIOLOGY: CHROMATIN DNA PACKAGING AND GENE SILENCING
The nucleosome is the basic repeat element of chromatin, and consists of 147 base pairs (bp) of DNA wrapped 1.7 times around an octamer of histone proteins (two copies each of the core histones H2A, H2B, H3, and H4).

Nucleosomes are connected by about 20 to 60 bp of linker DNA to form the 10-nm "beads-on-a-string" array. This can be further compacted into a "30-nm" chromatin fiber.

Two classes of model for chromatin have been proposed: (a) the "one-start helix" in which nucleosomes, connected by bent linker DNA, are arranged linearly in a higher order helix; or (b) the "two-start helix" in which nucleosomes, connected by straight linker DNA, zigzag back and forth between two adjacent helical stacks.

To distinguish between these two competing models of higher order chromatin folding, Dorigo and co-workers employed a fully defined in vitro system to generate regular nucleosomal arrays. Analysis of the length of the nucleosome stacks, now connected only by internucleosomal cross-links, revealed a two-start rather than a one-start organization. This interpretation was corroborated by electron microscopy. Thus, local interactions between nucleosomes can drive self-organization into a higher order chromatin fiber. Adapted from: Adone Mohd-Sarip and C. Peter Verrijzer (Science 2004 306:1484)1. B. Dorigo et al., Science 306, 1571 (2004)

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nucleotide

2007-12-17T23:08:00.000-08:00

Single chain (monomeric) nucleic acids are called nucleotides -- each consists of a nitrogenous heterocyclic base (either a purine or a pyrimidine), a pentose sugar, and a phosphate group.

More detail pending:
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nucleotide excision repair

2007-12-17T23:06:00.000-08:00

A nucleotide with abnormal alterations in its chemical properties is termed an elementary damage site (EDS). UV light is a frequent cause of damage to segments of DNA. Nucleotide excision repair is a multi-enzyme excision repair pathway targetted at damaged segments of DNA. Similar mechanisms exist in both prokaryotes and eukaryotes, and the mechanism is highly conserved in eukaryotes. The recognition of DNA damage is less specific in nucleotide excision repair (NER) than in base excision repair (BER), which targets damaged bases. Thus NER repairs a wider range of damage types than BER, irrespective of chromatin structure or the gene expression profile of a particular cell.

NER operates by: (a) recognition of the damaged DNA; (b) excision of an oligonucleotide of 24–32 residues by dual incision of the damaged strand on each side of the lesion; and, as for BER (c) filling in of the resulting gap by DNA polymerase; and (d) ligation of the nick. There is evidence that at least some steps of NER require ATP-dependent chromatin remodeling activities.

NER can operate by two pathways. The first pathway, global genome repair (GGR), acts on DNA lesions across the genome and is transcription-independent. The second NER pathway, transcription coupled repair, is coupled to active transcription and is directed to the transcribed strand of active genes.

clickable diagram - nucleotide excision repair : diagram>nucleotide excision repair : diagram>transition coupled repair : more detail NER defective medical disorders : free full text review article :

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nuclear speckles

2007-12-17T23:04:00.000-08:00

New Gene Regulation Mechanism Discovered: "Dr. David Spector noticed that under standard growth conditions, a particular population of messenger RNA molecules lingered in the nucleus indefinitely--in structures they call 'nuclear speckles'--and never reached the cytoplasm.

One of Spector's graduate students developed a method for purifying speckles. That allowed the researchers to identify not only the many different protein components of speckles, but also the messenger RNAs that are the basis of the new study, published in the October 21 issue of the journal Cell. The study--spearheaded by Cold Spring Harbor Laboratory postdoctoral fellow Dr. Kannanganattu Prasanth--identified the first such messenger RNA: one transcribed from a mouse gene called mCAT2 that encodes a cell surface receptor.

The scientists learned from the work of others that the mCAT2 receptor is involved in the production of nitric oxide, and that nitric oxide production is stimulated by various stress conditions including wound healing and viral infection. " That told us that when cells are stressed, maybe the atypical messenger RNA is released from the nucleus, exported to the cytoplasm, and translated into protein, thus circumventing the time-consuming process of producing new messenger RNA and providing a rapid response to viral infection or other stresses," says Spector. To test this idea, the researchers mimicked the effect of viral infection by treating cells with interferon.Sure enough, they discovered that the atypical mCAT2 messenger RNA in the nucleus was rapidly cleaved in response to interferon treatment, and that the protein coding portion of the molecule was then quickly exported to the cytoplasm and translated into protein. diagram.

"This 'cut and run' mechanism is a completely new paradigm of gene regulation, so studying it will keep us busy for a while. But we already suspect that there is going to be a large family of genes regulated in this way," says Spector.""

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open reading frame

2007-12-16T23:18:00.000-08:00

Because codons occur in nucleotide triplets, from any point within the genome there are six possible reading frames (three in each direction). Open reading frames (ORFs) are those sequences of DNA or RNA that code for translation at ribosomes into polypeptides or proteins. Within the genome, (ORFs) lie between codons for initiation (start-code sequence) and termination (stop-code sequence).

A DNA open reading frame starts with ATG—coding for Met—in most species, and ends with a stop codon (TAA, TAG, or TGA). In prokaryotes, ORFs are usually the longest sequence without a stop codon. In eukaryotes, long ORFs can continue over non-translatable intron gaps, so spliced mRNA must be employed to determine ORFs. Short ORFs can occur outside genes – within the intron, segments of DNA outside genes that were formerly considered ‘junk’ DNA.

More detail pending:
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oxidative stress and DNA damage

2007-12-16T23:04:00.001-08:00

Oxidative Stress/DNA Damage and DNA Repair: "Oxidative stress is produced in cells by oxygen-derived species resulting from cellular metabolism and from interaction with cells of exogenous sources such as carcinogenic compounds, redox-cycling drugs and ionizing radiations. DNA damage caused by oxygen-derived species including free radicals is the most frequent type encountered by aerobic cells. DNA damage caused by oxygen-derived species including free radicals is the most frequent type encountered by aerobic cells. When this type of damage occurs to DNA, it is called oxidative DNA damage and it can produce a multiplicity of modifications in DNA including base and sugar lesions, strand breaks, DNA-protein cross-links and base-free sites. "

MOLECULAR BIOLOGY: ON DNA-REPAIR ENZYMESThe agents that cause oxidative damage to DNA include oxygen radicals and ionizing radiation. Oxidation of a guanosine base to form oxoG produces a subtle structural transformation that results in deleterious mutations because DNA polymerases misread oxoG as a thymine (T) during genome replication prior to cell division. The human oxoG repair enzyme (hOGG1) catalyses the excision of oxoG in the first step of base excision repair. Structural studies of the glycosylase enzymes involved in the repair process reveal common features of damaged-base recognition. These include enzyme-initiated DNA distortion that flips the damaged base out from the DNA double helix for recognition within a base-specific cavity of the enzyme.(OGG1)

MOLECULAR BIOLOGY: ON DNA-REPAIR ENZYMES: "hOGG1 makes extensive contacts with the orphaned cytosine base, which ensures that oxoG is removed only when in the appropriate base-pairing context. Although extensive biophysical and structural studies intimate that there are general features of damaged bases that signal their presence to repair enzymes, the steps involved in finding damaged bases in a sea of normal ones are still unclear. Most mechanisms invoke the enzyme sliding or hopping along the DNA duplex until a damaged site is detected. A particularly intriguing question is whether normal bases are also extruded from the helix during the search process."

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oxoG repair

2007-12-16T23:04:00.000-08:00

MOLECULAR BIOLOGY: ON DNA-REPAIR ENZYMES
The agents that cause oxidative damage to DNA include oxygen radicals and ionizing radiation. Oxidation of a guanosine base to form oxoG produces a subtle structural transformation that results in deleterious mutations because DNA polymerases misread oxoG as a thymine (T) during genome replication prior to cell division. The human oxoG repair enzyme (hOGG1) catalyses the excision of oxoG in the first step of base excision repair.Structural studies of the glycosylase enzymes involved in the repair process reveal common features of damaged-base recognition. These include enzyme-initiated DNA distortion that flips the damaged base out from the DNA double helix for recognition within a base-specific cavity of the enzyme. (OGG1)

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polyadenylation

2007-12-15T23:21:00.000-08:00

Polyadenylation is a form of RNA processing in which the 3’ end of the pre-mRNA is cleaved before a stretch of adenosines are added to the end of the molecule. Employment of alternative polyadenylation sites can result in the insertion or deletion of sequences that control the stability of the mRNA, and thus the level of protein expression. The polyA tail is also involved in initiation of translation.

MOLECULAR BIOLOGY: ON TRANSCRIPTION TERMINATION: "A defining feature of mRNAs is a tail composed of a long string of adenosine nucleotides -- the poly(A) tail. This is not encoded by the gene but is added following cleavage of the nascent RNA transcript. Molecular factors that recognize the cleavage site, and cut the RNA, bind to a regulatory region of the transcribing RNA polymerase II called the carboxy-terminal domain (CTD). This interaction is important for recruiting the factors to the nascent transcript. In turn, these factors must be off-loaded from the polymerase onto the RNA at their site of action for transcription to be terminated(4,5). In fact, recognition of the cleavage site has been believed to be the key step in termination -- but the new data(1-3) show that this isn't the whole story."

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RNA processing

2007-12-15T23:20:00.000-08:00

Posttranscriptional RNA processing is an important feature of eukaryotic cells.

Pre-mRNA processing takes place in the nucleus. RNA processing events include capping of the 5’ end on the pre-mRNA, pre-mRNA splicing to remove intronic sequences, and polyadenylation of the 3’ end of the pre-mRNA.

Capping of the 5’ end of the nascent pre-mRNA is performed soon after initiation of transcription. Cleavage, pre-mRNA splicing, and polyadenylation usually follow termination of transcription of short primary transcripts with few introns. However, introns often are spliced out of the nascent RNA before transcription of the gene is complete for large genes with multiple introns.

diagram - pre-mRNA processing : animation of RNA splicing : animation - life cycle of an mRNA : diagram - spliceosome assembly : NCBI Molecular Cell Biology : Post-transcriptional Processing of RNAs

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pre-mRNA

2007-12-15T23:16:00.000-08:00

Precursor messenger RNAs, or heteronuclear RNAs (hnRNAs) are products of gene transcription found in the cell's nucleus, which contain bases complementary to those of the template DNA strand, including both exons and introns.

Following pre-mRNA processing, which includes capping, pre-mRNA splicing, alternative splicing, and polyadenylation, the mature mRNA is transported through nuclear pores into the cytoplasm.

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pre-mRNA splicing

2007-12-15T23:14:00.000-08:00

Because eukaryotic RNAs are transcribed from intron containing genes, the sequences encoded by the intronic DNA must be removed from the primary transcript prior to the RNAs becoming biologically active. The process of intron removal is called RNA splicing, or pre-mRNA splicing. The intron-exon junctions (splice sites) in the precursor mRNA (pre-mRNA) are recognized by trans-acting factors (prokaryote RNAs are mostly polycistronic). In pre-mRNA splicing the intronic sequences are excised and the exons are ligated to generate the spliced mRNA.

Group I introns occur in nuclear, mitochondrial and chloroplast rRNA genes, group II in mitochondrial and chloroplast mRNA genes. Many of the group I and group II introns are self-splicing in that no additional protein factors are necessary for the intron to be efficiently and accurately excised and the strands reattached.

Group I introns require an external guanosine nucleotide as a cofactor. The 3'-OH of the guanosine nucleotide acts as a nucleophile to attack the 5'-phosphate of the intron's 5' nucleotide. The 3' end of the 5' exon is termed the splice donor site. The 3'-OH at the 3' splice donor end of the 5' exon next attacks the splice acceptor site at the 5' nucleotide of the 3' exon, releasing the intron and covalently attaching the two exons together.

Pre-mRNA processing takes place in the nucleus of eukaryotes, whereas lack of a nuclear membrane in prokaryotes permits initiation of translation while transcription is not yet complete.Pre-mRNA processing events include capping of the 5’ end on the pre-mRNA, pre-mRNA splicing to remove intronic sequences, and polyadenylation of the 3’ end of the pre-mRNA.

animation of RNA splicing requires Flash Player plugin - Download plugin: clickable slide show - spliceosome intron removal :More in NCBI Molecular Cell Biology on-line text.

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proteome

2007-12-15T23:13:00.000-08:00

The term proteome refers to an organism’s collection of proteins. A cellular proteome is the collection of proteins found in the particular cell type under a particular set of environmental conditions. The complete proteome for an organism is the potential complete set of proteins, and equates to the sum of cellular proteomes under all possible conditions. The term, introduced in 1995, is analogous to the term genome for nucleic acids, though the proteins themselves are coded for by a portion of the genome.

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regulatory proteins

2007-12-13T23:47:00.000-08:00

Regulatory proteins bind to segments of DNA and bring about gene gene regulation.

More detail pending:
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replication

2007-12-13T23:45:00.000-08:00

Replication results in copies of the original DNA strand. First, the strands of the helix are unwound, then molecular machines move along each strand generating complementary strands, which then reassemble into a copy of the original.

Helicases are a critical part of the DNA replication process because they unwind double-stranded DNA to create single strands suitable for copying by the replication machinery. This and other helicase activity in the cell depends on the ability of the helicase's protein “engine” to crawl along the DNA strand. This locomotion is powered by ATP, the cell's ubiquitous energy source.

Helicase ProteinA helicase protein moving rapidly on a highly flexible single-stranded DNA track. Repetitive movement on the DNA may keep it clear of potentially toxic proteins. Watch Animation 8KB Flash Animation(requires Flash Player)

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ribosomes

2007-12-13T23:41:00.000-08:00

Ribosomes are large intracellular aggregates attached to the rough endoplasmic reticulum. They comprise several RNAs and scores of proteins, and function as ribozymes.

Ribosome biogenesis in eukaryotes mainly occurs in the nucleolus, a specialized nuclear compartment. The synthesis of ribosomal (rRNAs) is not achieved by simple transcription of the individual rRNA species, rather it requires a complex series of post-transcriptional processing steps.

Largest Computational Biology Simulation Mimics Life's Most Essential Nanomachine: "The ribosome is so fundamental to life that many portions of this molecular machine are identical in every organism ever genetically sequenced. In developing the project, the team identified a corridor inside the ribosome that the transfer RNA must pass through for the decoding to occur, and it appears to be constructed almost entirely of universal bases, implying that it is evolutionarily ancient." The original news release can be found here. Voxel simulation image : Image 2 : Image 3 : Image 4 : Image 5 : High res movie : Low res movie : See an image of the Q machine at http://www.lanl.gov/asci/.

ribosomal structure

2007-12-13T23:40:00.000-08:00

MOLECULAR BIOLOGY: ON RNA STRUCTURE
Elucidation of the crystal structures of the ribosome and its subunits have greatly increased the store of information about RNA structure. This information is moving toward an understanding of the principles of RNA folding and of the molecular interactions generating functional capabilities of the ribosome and other RNA systems. Almost all of the possible types of RNA tertiary interactions have been found in ribosomal RNA. One of these interactions is an abundant tertiary structural motif called the A-minor interaction. It has been observed that this participates in both aminoacyl-transfer RNA selection and in peptidyl transferase. It may also play an important role in the structural dynamics of the ribosome.

More detail pending:
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ribozymes

2007-12-13T23:39:00.000-08:00

Ribozyme Enzymology: "Ribozymes are antisense RNA molecules that have catalytic activity. They function by binding to the target RNA moiety through Watson-Crick base pairing and inactivate it by cleaving the phosphodiester backbone at a specific cutting site.Five classes of ribozymes have been described based on their unique characters in the sequences as well as three-dimensional structures (Bunnell,1997). They are denoted as (1) the Tetrahymena group I intron, (2) RNase P, (3) the hammerhead ribozyme, (4) the hairpin ribozyme, and (5) the hepatitis delta virus ribozyme. They may catalyze self-cleavage (intramolecular or 'in-cis' catalysis) as well as the cleavage of external substrates (intermolecular or 'in-trans' catalysis) (Ohkawa, 1995). "

Ribosomes are large intracellular aggregates attached to the endoplasmic reticulum. They comprise several RNAs and scores of proteins, and function as ribozymes.

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ribozymes in repair of RNA and DNA

2007-12-13T23:38:00.000-08:00

Ribozyme-mediated revision of RNA and DNA -- Long et al. 112 (3): 312 -- Journal of Clinical Investigation: modified: "Several classes of catalytic RNAs, or ribozymes, have the capacity to revise genetic information. Group I and Group II ribozymes are derived from naturally occurring Group I and Group II introns, respectively. These introns are found in the genes of a variety of lower eukaryotes and prokaryotes. They differ fundamentally from spliceosomal introns since Group I and Group II introns self-splice from the precursor RNA independent of the spliceosome. The intron adopts the catalytic structure that is capable of cleaving RNA splice sites and ligating the flanking exons together. In addition to self-splicing from RNA precursors, some Group II introns are able to reverse-splice into DNA. The splicing activity of these introns can be harnessed as a molecular tool that may potentially revise any gene of interest."

BIO.COM: Biotechnology & Pharmaceutical News, Jobs, Software, Reports, Books, Events: modified: "Hairpin ribozyme . . . minimal RNA enzyme was discovered by botanists in plants infected by tobacco ring spot virus and a number of other viruses. It is what is known as plant satellite RNA.Satellite RNAs are parasitic pieces of RNA that are not exactly viruses because they don't encode for proteins. Instead, they catalyze simple cut-and-paste reactions in order to replicate themselves, exacerbating or ameliorating diseases caused by plant viruses. A plant that has tobacco ring spot virus, for instance, will be more diseased if it also has this satellite RNA. "

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RNA

2007-12-13T23:17:00.000-08:00

RNA differs from DNA in its general composition -- ribose is the sugar moiety in the nucleotide backbone, and uracil serves as a base.

In general, a cell has more RNA than DNA, mostly incorporated in ribosomes with a relatively low rate of turnover. Only a small portion of human DNA has coding potential. Almost all RNA sequences in the cytoplasm, being derived from DNA coding sequences, have functional significance, whether transcribed in coding for proteins (mRNA), in performing translation (ribosomal RNA as ribozymes, and tRNA), or in epigenetic mechanisms. Substantial damage to RNA induces apoptosis (cell death), so RNA repair mechanisms are vital to cells.

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RNA polymerase

2007-12-13T23:16:00.000-08:00

In prokaryotic cells, all RNA classes are synthesized by a single polymerase. In eukaryotic cells there are 3 distinct classes of RNA polymerase, RNA polymerase (pol) I, II and III. Each polymerase is responsible for the synthesis of a different class of RNA.

RNA polymerase I (Pol I) is dedicated to synthesis of pre-rRNA. RNA polymerase II (Pol II) initiates transcription at DNA sequences corresponding to the 5 Cap of mRNAs and transcribes pre-mRNA. RNA polymerase III (Pol III) transcribes tRNA genes, 5S-rRNA genes, and genes encoding several other small RNAs.

In order to begin transcription, RNA polymerase requires a number of general transcription factors (called TFIIA, TFIIB, and so on). The general transcription factors have been highly conserved in evolution.

All RNA polymerases are dependent upon a DNA template from which to synthesize RNA by addition of complementary bases to an elongating backbone. In RNA, U is substituted for T. The transcribed RNA is complementary to the template strand of the DNA duplex, and is a replica of base-sequences in the non-template, coding strand. The non-template strand is called the coding strand because its sequences are identical to those of the mRNA (with U substituted for T).

p53 - A 3D animation showing the molecule p53 binds to DNA and initiates the transcription of mRNA : diagram - pre-mRNA processing : diagram - intron excision in mRNA precursors : life cycle of an mRNA ~ click on Quicktime Q : alternative splicing - click on fig 1 for animation :NCBI Molecular Cell Biology - Transcription Initiation Complex : Bacterial Transcription Initiation (NCBI MCB) : SUMMARY transcription initiation (NCBI MCB)

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messenger RNA

2007-12-13T23:14:00.000-08:00

Messenger RNA, abbreviated mRNA, results from transcription of the base sequence of a segment of single strand archival DNA in the cell's nucleus.

In eukaryotic cells, mRNAs are transcribed as pre-mRNA and processed into mature mRNA in the nucleus, then transported through nuclear pore complexes to the cytoplasm. In some cases, mRNAs are transported to specific areas of the cytoplasm before being translated by ribosomes.

Complex macromolecular machines, such as spliceosomes and the cleavage/ polyadenylation apparatus, control each of these fundamental processes. These macromolecular assemblages comprise scores of proteins, and in many cases RNAs. The complexity of these control-assemblages ensures accuracy in locating promoters and splice sites in the long length of DNA and RNA sequences. Further, the macromolecular machines provide various avenues for regulating synthesis of a polypeptide chain.

Regulation of mRNA stability is central to the post-transcriptional modulation of gene expression. Stability of mRNA varies considerably between mRNAs and can be modulated by extracellular stimuli. Tight control of mRNA stability permits rapid changes mRNA levels, providing a mechanism for prompt termination of protein production. The rate of mRNA decay is determined by cis-acting sequences within the mRNA, which are recognized by trans-acting factors. The best-characterized cis-acting sequences responsible for mRNA decay in mammalian cells are the AU-rich elements, AREs present within the 3'-UTRs of short-lived mRNAs. These AREs are involved in deadenylation and subsequent degradation of mRNAs, and they have also been observed to stimulate 5'-decapping.

Dysregulation of mRNA stability has been associated with different chronic inflammatory diseases, -thalassemia, cancer, and Alzheimer's disease.A number of ARE binding proteins (ARE-bps) have been identified, which interact with AU- and U-rich regions. These include the ELAV protein family members (most important HuR), the ARE/poly-(U)-binding/degradation factor 1 (AUF-1, also named hnRNP D), the heteronuclear ribonucleoprotein A1 (hnRNP A1), the KH-type splicing regulatory protein (KSRP), tristetraprolin (TTP), the T cell-restricted intracellular antigen (TIA)-1 and the TIA-related protein (TIAR). The KH-type splicing regulatory protein, KSRP has been described as being essential for exosome-mediated degradation of ARE-containing mRNAs.

Full Text Research Article on KSRP-mediated reduction of iNOS expression.animation - life cycle of an mRNA : animation ~ alternative splicing : NCBI Molecular Cell Biology - Contents : Google mRNA : Classes of RNA Polymerases : Mechanism of RNA Polymerases : Processes of Transcription : Post-transcriptional Processing of RNAs : RNA Splicing : Clinical Significance of Alternative and Aberrant Splicing

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miRNAs

2007-12-13T23:13:00.000-08:00

microRNAs are a large class of 21 to 24 nucleotide non-coding RNAs that have probable regulatory roles in animals and plants.

More detail pending:
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tRNA

2007-12-13T23:12:00.000-08:00

Largest Computational Biology Simulation Mimics Life's Most Essential Nanomachine: "The simulations also reveal that the essential translating molecule, transfer RNA, must be flexible in two places for decoding to occur, furthering the growing belief that transfer RNA is a major player in the machine-like movement of the ribosome. The simulation also sets the stage for future biochemical research into decoding by identifying 20 universally conserved ribosomal bases important for accommodation, as well as a new structural gate, which may act as a control mechanism during transfer RNA selection."

The original news release can be found here. : Voxel simulation image : Image 2 : Image 3 : Image 4 : Image 5 : High res movie : Low res movie : See an image of the Q machine at http://www.lanl.gov/asci/.

More detail pending:
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silencers

2007-12-12T23:37:00.000-08:00

Silencers are control sections of DNA that, like enhancers, may be located thousands of base pairs away from the gene they regulate. However, when transcription factors bind to them, expression of the gene they regulate is repressed. This is the opposite effect to that of enhancers, which increase rate of transcription.

MOLECULAR BIOLOGY: CHROMATIN DNA PACKAGING AND GENE SILENCING:"One basic premise of chromatin regulation is that genes are silenced through compaction of chromatin, which reduces the accessibility of DNA. In contrast, gene expression may require the "opening up" of chromatin. The Polycomb group (PcG) of gene repressors and the trithorax group (trxG) of gene activators are two antagonistic classes of proteins that may act through modulation of chromatin structure. Together, these factors maintain the gene expression patterns of key developmental regulators and hence are crucial players in cellular differentiation, stem cell renewal, and cancer."

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snoRNAs

2007-12-12T23:34:00.000-08:00

Small nucleolar RNAs

More detail pending:
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spliceosome

2007-12-12T23:18:00.000-08:00

Almost all eukaryotic protein-coding genomes contain non-coding intervening sequences called introns. Spliceosomes are complex ribonuclear machines in eukaryotes that remove the non-coding introns from primary transcript, precursor mRNA (pre-mRNA or hnRNA). Alternatively, the term spliceome can be used to describe the complete set of all possible alternative splices in an organism, analogous to the genome or proteome.

Spliceosomes are variably composed of as many as 300 distinct proteins and five RNAs, making them among the most complex macromolecular machines known. review article abstract. Essential components of the spliceosome are the small RNA-protein complexes called small nuclear ribonucleoproteins (snRNPs, pronounced 'snurps'). These are U1, U2, U4, U5, and U6, so named because they are rich in uridine nucleotides. In addition to snRNPs, splicing requires many non-snRNP protein factors. The snRNPs participate in several RNA-RNA and RNA-protein interactions.

The spliceosome recognizes specific 5' and 3' sites on the pre-mRNA. The intronal area between these locations is excised, and the two exons are spliced (ligated).

diagram - formation of a spliceosome : diagram - intron excision in mRNA precursors : diagram - pre-mRNA processing : animation ~ alternative splicing : animation of RNA splicing requires Flash Player plugin - Download plugin: clickable slide show - spliceosome intron removal : alternative splicing - click on fig 1 for animation : life cycle of an mRNA ~ click on Quicktime Q : clickable slide show - spliceosome intron removal :

Aberrant splicing creates mutant proteins, while alternative splicing generates diversity. Splice variants and epigenetic mechanisms account for the ability of about 25,000 human genes to code for about 100,000 human proteins. In addition to this variation, and that provided by recombination, each human gene possesses at least two isoforms – one from each parent. X Inactivation

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SSOs

2007-12-12T23:12:00.000-08:00

Increased efficiency of oligonucleotide-mediated gene repair through slowing replication fork progression -- Wu et al. 102 (7): 2508 -- Proceedings of the National Academy of Sciences: "In mammalian cells, the precise mechanism of SSO-mediated chromosome alteration remains to be established, and there have been problems in obtaining reproducible targeting efficiencies. It has previously been suggested that the chromatin structure, which changes throughout the cell cycle, may be a key factor underlying these variations in efficiency. "

More detail pending:
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trans-splicing ribozymes and therapeutics

2007-12-11T23:15:00.000-08:00

Ribozyme-mediated revision of RNA and DNA -- Long et al. 112 (3): 312 -- Journal of Clinical Investigation: "The therapeutic revision of RNA by ribozymes is mechanistically different from that of DNA. To revise RNA sequences, a Group I intron from Tetrahymena thermophila has been adapted for trans-splicing. Trans-splicing ribozymes splice therapeutic RNA sequences onto a target transcript in a process called ribozyme-mediated RNA repair. Considerable progress has been made in the development of trans-splicing ribozymes for therapeutic applications such as treating sickle cell disease and cancer." This is one of the relatively recent targeted genetic repair techniques.

"The spliceosome has also been shown to trans-splice RNA transcripts. Spliceosomal-mediated RNA trans-splicing is also called called SMaRT."

More detail pending:
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termination of transcription

2007-12-11T23:10:00.000-08:00

MOLECULAR BIOLOGY: ON TRANSCRIPTION TERMINATION: "Transcription termination is important, not least because stopping too late will disrupt the regulation of other genes on the same chromosome; stopping prematurely, meanwhile, would produce truncated and therefore defective mRNAs. . . termination on most genes has been found to occur at various positions rather than at a single site. Moreover, a discrete signal in the DNA that might define the termination site could not be identified. . . Recent work(1-3) address this problem and provides evidence in favor of a striking alternative mechanism: that the transcribing RNA polymerase is "torpedoed". The mRNA is cut while it is still being synthesized, with the liberated region forming the mRNA. The remaining RNA trails out of the transcribing RNA polymerase, and is attacked by an exonuclease -- an enzyme that can degrade the RNA from its free end. This enzyme chases after the polymerase, chewing up the RNA strand as it goes. When it catches up with the polymerase, transcription is terminated."

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translation

2007-12-11T23:09:00.000-08:00

Translation is the process by which messenger RNAs derived from archival DNA are translated into polypeptides and proteins at ribosomes.

More detail pending:
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Research techniques

2007-12-01T00:00:00.000-08:00

Computer Simulation of Ribosome at LANL :
exogenous nucleobase rescue of abasic substitutions siRNA :
SMaRT Spliceosome-Mediated RNA Trans-splicing :
E-MAP Technique Offers New View of Dynamic Biological Landscape :

More detail pending:
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2007-11-30T23:59:00.000-08:00

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2007-11-14T13:12:00.000-08:00

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STANFORD RESEARCHERS DEVELOP SYSTEM FOR FIELD TESTING MECHANISMS OF EVOLUTION

2006-05-07T11:16:00.000-07:00

STANFORD RESEARCHERS DEVELOP SYSTEM FOR FIELD TESTING MECHANISMS OF EVOLUTION: "Kingsley said they found a number of parallels between traditional laboratory genetics and the traits they examined in the stickleback populations. For example, many of the traits could be traced to major chromosome regions - indicating that evolution can occur through changes of large effect, not just as a series of small changes. Their findings also indicate that genetic control of body regions appears to be modular. The genes that control the length of the first dorsal spine, for instance, are located on different chromosome regions from the genes that control the
length of the second dorsal spine. This is not surprising, said Kingsley, because it follows previous findings of the genetic control of mouse skeleton development. As anyone who plays with Legos can testify to, a modular body plan greatly increases the options for tweaking designs over time."

ScienceDaily: Groovy Protein Essential For Promoting Cancer Development

2006-02-06T13:55:00.002-08:00

ScienceDaily: Groovy Protein Essential For Promoting Cancer Development: "Cancer researchers have long sought a way to subdue telomerase, an enzyme whose excessive activity contributes to the unchecked growth of as many as 90 percent of human tumors. The enzyme is vital for some rapidly dividing cells -- such as those in a developing embryo -- where it extends telomeres, the regions of highly repetitive DNA found at the ends of chromosomes. In most healthy adult cells, telomerase is shut off, and telomeres slowly shrink during cell division -- a normal process that helps limit cells' lifespan. Cancer cells, however, usually find a way to turn telomerase back on, achieving a dangerous immortality. "

Genome Sequencing Is For Ecologists, Too

2006-01-25T15:00:00.000-08:00

Genome Sequencing Is For Ecologists, Too: "Daphnia's short generation time and small genome (a mere 200 million base pairs) makes it an ideal organism for laboratory and field studies of how environments influence -- and how they're influenced by -- an organism's genetics. The animals are common in lakes and ponds and have been used to monitor the health of aquatic environments. Members of the species can reproduce both with and without sex, which has important implications in evolutionary biology and ecology."

New Cellular Flaw Found In Some Virulent Breast Cancers

2006-01-23T13:13:00.000-08:00

New Cellular Flaw Found In Some Virulent Breast Cancers: "Breast cancers composed of cells that contain both the overactive cyclin D1- CDK4 switch and a mutated cancer-causing gene ErbB-2 (also known as HER2) are extremely difficult to treat. In one recent study, the seven-year survival rate for women with this subgroup of breast cancers was only about 13 percent. "

Researchers Discover How A High-fat Diet Causes Type 2 Diabetes

2005-12-29T10:55:00.000-08:00

Researchers Discover How A High-fat Diet Causes Type 2 Diabetes: "In an article published in the December 29, 2005, issue of the journal Cell, the researchers report that knocking out a single gene encoding the enzyme GnT-4a glycosyltransferase (GnT-4a ) disrupts insulin production. Importantly, the scientists showed that a high-fat diet suppresses the activity of GnT-4a and leads to type 2 diabetes due to failure of the pancreatic beta cells. "

Noise and Delays Explain Why Some Genes Oscillate in Activity

2005-12-28T10:10:00.000-08:00

Noise and Delays Explain Why Some Genes Oscillate in Activity [News & Events]: "In a paper in Proceedings of the National Academy of Sciences released online Sept. 30, the scientists led by bioengineering professor Jeff Hasty and research scientist Lev Tsimring reported that unscripted biochemical variations, or noise, combined with time delays in certain biochemical reactions may lead to oscillations in gene regulation that couldn�t otherwise be predicted. Such noise is routinely described by cell biologists who record large phenotypic differences between supposedly identical cells in a single flask of growth medium. "

Aspergillus fumigatus genome

2005-12-28T10:02:00.000-08:00

What's New: "Unlike most fungi, A. fumigatus likes it hot--and hotter. The fungus enjoys an unusual range of temperatures. At home in the compost heap, A. fumigatus tolerates temperatures up to 70 degrees Celsius. The fungus becomes a human pathogen because it's perfectly comfortable at body temperature, 37 degrees C. Altering ambient temperatures in the lab, TIGR scientists tracked gene activity, documenting different A. fumigatus genes that turned on and off, as the environment warmed.
The A. fumigatus genome is 28 Mb in size, consisting of 8 chromosomes bearing a total of almost 10,000 genes. Which genes make the mold virulent? Some 700 A. fumigatus genes significantly differ--or do not even occur--in a similar, yet less infectious fungus, Neosartorya fischeri. Nierman and colleagues are now searching these unique genes for clues to A. fumigatus infectivity.
It's a complex task. Suspect genes encode proteins involved in central metabolic pathways, cell signaling, cell wall biosynthesis, pigment biosynthesis, and secondary metabolite production. In other words, A. fumigatus's virulence genes are likely complex and mixed up with normal metabolic capabilities, Nierman says. He and his colleagues now plan to systematically 'knock out,' or disable, genes that might make A. fumigatus infectious. Eventually, Nierman adds, this work could lead to better therapies for serious asthma, allergy, and other conditions."

Researchers Quantify More Noise In Gene Expression

2005-12-28T09:52:00.000-08:00

Researchers Quantify More Noise In Gene Expression: ""Many individual genes produce less than 10 copies of regulatory messenger molecules, which is such a small number that it makes clockwork-like regularity of downstream cellular circuits statistically impossible. Our group and others have detected and classified significant downstream fluctuations that result from this source of 'intrinsic noise' in gene expression"

Hasty leads a team of researchers at UCSD that will report in a Dec. 21 advanced online publication by Nature a mathematical description of “extrinsic noise,” an even larger component of variation in gene expression. This second type of noise results because no two genetically identical cells can keep the same time. The measurement of extrinsic noise was based on experiments involving baker's yeast, Saccharomyces cerevisiae, but the phenomenon and the way it is described mathematically would apply to other types of cells and other species.

Intrinsic noise resulting from low copy numbers of molecules has been the focus of earlier studies by Hasty’s group. Its Nature paper focuses on extrinsic noise, an even larger source of variation between identical cells growing side-by-side in identical conditions. “This extrinsic noise originates from cells being out of phase in their growth cycle,” said Hasty. “It’s unavoidable. This study also established an ‘extrinsic-noise floor’ in 11 genes for which intrinsic noise is negligible.”
The group measured the noise floor by used genetically engineered strains of yeast with a green fluorescence marker linked to a gene involved in the metabolism of the sugar galactose. The galactose gene and 10 others were chosen because they are expressed in yeast cells in high copy numbers, a feature that makes intrinsic noise in their expression statistically insignificant. By making cell-to-cell comparisons of the activation of the galactose-fluorescence gene pair in experiments with one to five copies of the gene pair per cell, the team was able to measure the extrinsic-noise floor, a parameter that is needed to create a mathematical algorithm describing gene-expression variation in yeast.
“This may sound relatively straightforward: extrinsic variability is due to individual cells growing out of phase with one another,” said Hasty. “However, this study is the first rigorous description of this variability, which will be very helpful to researchers eager to account for variations in gene expression as they build rigorous, comprehensive models of this and other types of cells.”"

Dimitri Volfson, Jennifer Marciniak, William J. Blake, Natalie Ostroff, Lev S. Tsimring, and Jeff Hasty, "Origins of extrinsic variability in eukaryotic gene expression" (2005). Nature. doi: 10.1038/nature04281.

New Study Shows Animal Family Tree Looking Bushy In Places

2005-12-26T11:53:00.000-08:00

New Study Shows Animal Family Tree Looking Bushy In Places: "'It turns out that early in the origin of many types of animals, there were a lot of branching events in a short period of time,' Carroll explains. Those type of episodes at key junctures in life's history - for example, the rise of complex animals or the migration of vertebrates from the sea to land - make the animal tree look very bushy and very murky, Carroll's group reports."

MicroRNA Gene That Regulates Lifespan Found By Yale Scientists

2005-12-26T10:54:00.000-08:00

MicroRNA Gene That Regulates Lifespan Found By Yale Scientists: ""Although there is a large variation in lifespan from species to species, there are genetic aspects to the processes of development and aging," said Frank Slack, associate professor of Molecular, Cellular and Developmental Biology and senior author of the paper. "We used the simple, but genetically well-studied, C. elegans worm and found genes that are directly involved in determination of lifespan. Humans have genes that are nearly identical."

A microRNA and the developmental-timing gene it controls, lin-4 and lin-14, affect patterns of cellular development at very specific stages. Slack’s group found that mutations in these genes alter both the timing of the worm development stages— and the worm lifespan. C. elegans has been the premier model organism for studying the genetics of aging, and an excellent predictor of genes that also control mammalian aging."

New View Of Cancer: 'Epigenetic' Changes Come Before Mutations

2005-12-23T09:11:00.000-08:00

New View Of Cancer: 'Epigenetic' Changes Come Before Mutations: "Epigenetic changes -- those that don't affect the gene's sequence of DNA but change the gene in other ways -- influence a wide variety of human diseases, including cancer, birth defects and psychiatric conditions. Epigenetic alterations include the turning off or quieting of genes that normally suppress cancer and the turning on of oncogenes to produce proteins that set off malignant behavior.

Feinberg and his colleagues propose that cancers develop via a three-step process. First, there is an epigenetic disruption of progenitor cells within an organ or tissue, altered by abnormal regulation of tumor-progenitor genes. This leads to a population of cells ready to cause new growth.

The second step involves an initiating mutation within the population of epigenetically disrupted progenitor cells at the earliest stages of new cell growth, such as the rearrangement of chromosomes in the development of leukemia. This mutation normally has been considered the first step in cancer development.

The third step is genetic and epigenetic instability, which leads to increased tumor evolution.

Many of the properties of advanced tumors, including the ability to spread, or metastasize, are inherent properties of the progenitor cells that give rise to the primary tumor, Feinberg notes. These properties do not necessarily require other mutations to occur. "

Novel Enzyme Offers New Look At Gene Regulation

2005-12-21T15:31:00.000-08:00

Novel Enzyme Offers New Look At Gene Regulation: "The first enzyme to remove methyl groups from histones, or histone demethylase, was identified last year. This was a breakthrough in the study of histone modifications, but Zhang thought pieces of the puzzle were still missing.
'We hypothesized that there were more demethylase enzymes out there for two reasons,' Zhang said. 'For one, the previous demethylase identified, called LSD1, could not remove a chain of three methyl groups from a histone, or a trimethyl group. Secondly, common baker's yeast does not have LSD1, although it does have proteins adding methyl groups to histones.'"

Gene Mutation Found That Increases Severity Of Multisystem Syndrome

2005-12-20T08:29:00.000-08:00

Gene Mutation Found That Increases Severity Of Multisystem Syndrome: "'Scientists are going to have to think very hard before they discount genetic variation that appears not to directly cause a disease,' says the study's leader, Nicholas Katsanis, Ph.D., associate professor in the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins. 'The onus is on us to figure out how to dissect the effects of what appear to be silent genetic variants. I have a greatly renewed respect for the complexity of the genome, for the subtle ways that genes and gene products interact with each other.'"

Chromosome Four Contains Genes That Affect Drinking Behaviors In Smokers

2005-12-20T08:26:00.000-08:00

Chromosome Four Contains Genes That Affect Drinking Behaviors In Smokers: "A recent examination of families selected for their smoking behavior has identified the same region of chromosome four that was identified by earlier studies as being linked to the initiation of alcohol consumption. Results are published in the December issue of Alcoholism: Clinical & Experimental Research."

Mayo Clinic Discovers Two Key Players In Cancer Prevention And How They Work

2005-12-20T07:31:00.000-08:00

Mayo Clinic Discovers Two Key Players In Cancer Prevention And How They Work: "These new cancer players comprise a two-protein complex Rae1-Nup98. This complex works together to stabilize healthy cells by functioning as a kind of cell-division auditing system that makes sure the right number of chromosomes is distributed in each part of a newly divided cell. By so doing, they promote 'euploidy' -- the ideal chromosomal distribution needed for stability, and the opposite of aneuploidy.

"What we discovered is that there's an active process of cellular machinery that prevents aneuploidy," says Mayo cancer researcher Jan van Deursen, Ph.D., who led the research team. "It's a surveillance mechanism involving the two proteins Rae1-Nup98 that makes sure that in every cell division the proper number of chromosomes occur.""

Reconstructive Approach to Genetic Circuits

2005-12-17T14:54:00.000-08:00

SYSTEMS BIOLOGY: ON RECONSTRUCTION OF GENETIC CIRCUITS: "A reconstructive approach to genetic circuits may offer unique insight into their underlying mechanisms. In this approach, one constructs synthetic replicas of natural genetic circuits out of well-characterized elements, such as genes, proteins, regulatory sequences, and so on, and observes their dynamics in living cells. This scheme offers several advantages. First, one can test the sufficiency of an arbitrary circuit for generating a particular function. Second, one may study the circuit mechanism without impairing cellular functions or inducing downstream consequences. Third, different circuit designs with similar functions can be directly compared to determine their relative advantages and disadvantages. "

Rules To Target RNA Are Focus Of Research

2005-12-16T21:16:00.000-08:00

Rules To Target RNA Are Focus Of Research: "Rationally designed RNA inhibitors could, he explained, prove more valuable than molecules that inhibit DNA.
One reason is that while DNA bases or nucleotides are always paired according to the same formula, RNA bases have more diverse pairings, which makes targeting RNA more challenging, but also potentially more valuable.
'The ability to form different pairings allows RNA to have a much larger structural repertoire than DNA and that gives RNA the ability to have such diverse cellular functions,' said Disney.
In addition, he said, because DNA is present only in the nucleus, pharmaceutical compounds that target it must be able to penetrate the nucleus.
'Since RNA is present both in the cell's nucleus and cytoplasm, you do not need to get a compound into the nucleus to target it,' he said.
Because RNA folds more like a protein than DNA does, it also may be easier to design compounds that selectively target specific structures, he added."

E. Coli Bacterium Generates Simplicity From Complexity

2005-12-16T08:05:00.000-08:00

How E. Coli Bacterium Generates Simplicity From Complexity: "?This study gives a systems biology view of how a phenotype, or a ?network state? advantageous to a microorganism is comprised of a tiny subset of a much larger universe of possibilities as provided for in the genome,? said Palsson. ?On a high level we can say that E. coli is obsessed with how it breathes and whether or not glucose is available to eat. All of its genetic complexity basically enables it to generate a nice steady state for itself regardless of what it has to live on.?"

Fishing for the Origins of Genome Complexity

2005-12-16T07:55:00.000-08:00

Georgia Institute of Technology :: News Room :: Fishing for the Origins of Genome Complexity: "Biologists at Georgia Tech have provided scientific support for a controversial hypothesis that has divided the fields of evolutionary genomics and evolutionary developmental biology, popularly known as evo devo, for two years. Appearing in the December 2005 issue of Trends in Genetics, researchers find that the size and complexity of a species' genome is not an evolutionary adaptation per se, but can result as simply a consequence of a reduction in a species' effective population size.

There are some species of frogs and some amoeba that have much larger genomes than humans. To help explain this paradox, a pair of scientists from Indiana University and the University of Oregon published a hotly-contested hypothesis in 2003. It said that most of the mutations that arise in organisms are not advantageous and that the smaller a species effective population size (the number of individuals who contribute genes to the next generation), the larger the genome will be.

Their test consisted of analyzing data from 1,043 species of fresh and saltwater ray-finned fish. Previous data on genetic variability had established that freshwater species have a smaller effective population size than their marine counterparts. If the hypothesis was correct, the genome size of these freshwater fish would be larger than that of the saltwater dwellers. It was.

Then they matched the data with estimates of heterozygosity, a measure of the genetic variation of a population. Again they found that species with a smaller effective population had larger genomes.

?We see a very strong negative linear relationship between genome size and the effective population size,? said Soojin Yi, assistant professor in the School of Biology and lead author of the study. ?This observation tells us that the mutations that increase the genome tend to be slightly deleterious, because population genetic theories predict such a relationship.?

?The interesting thing here is that biological complexity may passively evolve,? said Yi. ?We show that at the origins, it?s not adaptive mutations, but slightly bad ones that make the genome larger. But if you have a large genome, there is more genetic material to play with to make something useful. At first, maybe these mutations aren?t so good for your genome, but as they accumulate and conditions change through evolution, they could become more complex and more beneficial.? "

Call for a Large-scale Human Epigenome Project

2005-12-16T07:47:00.000-08:00

: "Epigenetic modifications to DNA exert profound influences on gene activity. For example, studies suggest that epigenetic variation may be responsible for subtle differences in appearance and behavior of identical twins, whose gene activity profiles at 3 years of age are nearly alike, but by age 50 diverge as much as unrelated individuals in the population at large.

Epigenetic modifications take several forms. The most intensively studied have been the addition of methyl groups, small “beads” of carbon and hydrogen, to DNA, which generally correlate with low gene activity. The histone proteins – molecular “clips” that hold the six feet of DNA tightly wound inside each cell - are modified by methylation, but also by the addition of chemical entities containing acetic acid, phosphorus, and a number of other species.

The complex nature of epigenetic modifications constitutes a challenge to the development of reliable, high-throughput methods of cataloging them. Several workshop participants, however, working in both industrial and academic settings, have developed techniques proving adept at tackling epigenetic complexity.

These include the so-called ChIP/chip methodology, in which intact chromatin – the complex of DNA and histones – is immunoprecipitated (brought out of solution using antibodies that recognize specific histone modifications) and analyzed on microarray “chips.” Modifications of DNA are also tracked on chips, following treatment with enzymes that recognize sites of methylation."

Progeria: Mutant Lamins Cause Premature Aging

2005-12-14T13:28:00.000-08:00

Zeroing In On Progeria: How Mutant Lamins Cause Premature Aging: "Progeria researchers made a breakthrough in 2003, tracing HGPS to a spontaneous mutation in a gene encoding an important structural component of the cell nucleus, the organelle in which our DNA is stored, read out, and copied.

As the so-called "Mothership of the Human Genome," the cell nucleus must keep all this vital genetic information safe but accessible inside a strong protective envelope. The inner membrane of the nuclear envelope is lined by tough but adaptable proteins called lamins. The mutated gene for HGPS affected the nuclear lamin A (LA) protein.

The discovery that progeria was a "laminopathy," a disorder caused by a nuclear lamin failure, gave HGPS families new hope because it gave clinical researchers new targets for drug or other interventions. But the discovery gave cell biologists a new problem. If HGPS was cellular aging run wild, was it a warp-speed version of "normal" aging? If so, what was it about the mutated LA protein behind HGPS that causes cells to age so rapidly?

While lamins polymerize into fibrous structures that hold up the "walls" of the nucleus, they also serve as an internal scaffold for the complex machinery involved in DNA replication and gene expression. It was in this later role that the researchers have been looking for clues to premature and possibly to normal aging.

the mutant LA protein seems to interfere with key controls of gene expression and of the cell cycle. The first study discovered that the most common HGPS-linked mutant LA protein alters the organization of regions of chromosomes that are critically important in regulating gene expression. These so-called heterochromatic regions include the inactive X (Xi) chromosome found in normal female cells. One of the hallmarks of Xi heterochromatin is its association with proteins known as methylated histones. In the cells from a female HGPS patient, the researchers found that levels of this molecular hallmark and of an enzyme required for histone methylation of Xi are sharply lower.

The second set of results reveals mutant LA proteins turning up in the wrong place--too tightly linked to the membranes of the nuclear envelope--to be of much help during key stages of the cell cycle. The researchers believe that this localization failure of mutated LA proteins would severely compromise the ability of HGPS cells to engage in normal DNA replication, a probable factor in their rapid march to premature senescence. Whether similar missteps and miscues by nuclear lamins are part of "normal" human aging is the question that draws researchers onward, says Goldman."

Strep mutans survive without SRP (signal-recognition pathway)

2005-12-13T17:49:00.000-08:00

Bacteria That Cause Tooth Decay Able To Survive Without Important Biochemical Pathway: "Scientists have long believed a certain biochemical pathway involved in the folding and delivery of proteins to cell membranes is essential for survival. Now University of Florida researchers have discovered that Streptococcus mutans, the decay-causing organism that thrives in many a mouth, can do just fine without it."

ESTs

2005-12-13T17:36:00.000-08:00

Flatworm Genes May Provide Insights Into Human Diseases, Researchers Say: "ESTs are short sequences of DNA produced by the reverse transcription of messenger RNA into complementary DNA. Sequencing and categorizing ESTs allow researchers to rapidly identify genes. Previously sequenced ESTs came from asexual planarians.

"One of the striking things we found is that when we look at planarian genes, we see a group that is conserved between planarians and mammals that is not found in Drosophila or C. elegans," said Phillip A. Newmark, a professor of cell and developmental biology at Illinois. "We speculate that these conserved sequences may play roles in processes such as long-term tissue maintenance and cell turnover that are likely less important for short-lived organisms like nematodes and insects," wrote Newmark and colleagues."

What To Sequence Next: Pick One Species At A Time

2005-12-10T15:03:00.000-08:00

What To Sequence Next: Pick One Species At A Time: "DNA sequencing has revealed a vast amount of information about biology. But genome sequencing remains expensive and time consuming, so scientists need a strategy to help them select the organisms that will give them the most new information.

One solution is to sequence the most distantly related organisms, to get the widest possible diversity of sequences. Biologists represent the relationships between different species as a tree, with the length of the branches varying according to the degree by which their DNA sequences differ. "If we are prepared to assume that the most informative set is the one with the greatest evolutionary divergence, the problem of which species to sequence next can be solved by observing the length of the branches that separate the unsequenced species from those that have already had their genomes sequenced, and choosing the organism that's separated from the others by the longest sequence of branches", explains Fabio Pardi.

The tendency has been for centres to choose a group of new genomes to sequence. However, the current study shows that picking the best candidates one at a time is equally informative. "Computer scientists call this a 'greedy strategy' because it involves always taking the best bet for yourself", says Nick Goldman. "However, if, say, a centre had enough funding to sequence five organisms, we might expect to get a better set of genomes by considering all five together. Counterintuitively, we found that in this case the greedy strategy is the best. We were surprised because in computer science greed is definitely not good – greedy algorithms seldom provide the best solution to a problem."

"Our findings have clear implications for planning large-scale genome sequencing efforts", continues Pardi. "Provided that they remain open about their choices so that two different sequencing centres don't choose the same genome, selecting the next most attractive organism to sequence is just as effective as having a long-term strategy."

Evolutionary divergence isn't the only factor that scientists consider when choosing which genomes to sequence, but other criteria can be factored into Goldman and Pardi's greedy strategy so long as those criteria can be quantified. For example, sequencing costs, or the economic importance of an organism, could be considered. Their strategy can also be applied to different problems, such as conservation biology. "Of course, we're not advocating that genome scientists or conservation biologists stop working cooperatively, but at least they can feel confident about sequencing or conserving the organism of their choice without messing things up for their collaborators," says Goldman.""

Dog Genome Sequence; Analysis Sheds Light On Human Disease; Differences Among Canine Breeds

2005-12-08T07:57:00.000-08:00

Researchers Publish Dog Genome Sequence; Analysis Sheds Light On Human Disease; Differences Among Canine Breeds: "While dogs occupy a special place in human hearts, they also sit at a key branch point, relative to humans, in the evolutionary tree. It was already known that humans share more of their ancestral DNA with dogs than with mice; the availability of the dog genome sequence has allowed researchers to describe a common set of genetic elements -- representing about 5 percent of the human genome -- that are preferentially preserved among human, dog and mouse. Rather than being evenly distributed, some of these elements are crowded around just a small fraction of the genes in the genome. Future studies of these clusters may give scientists the critical insight needed to unravel how genomes work.

The international team of researchers also identified roughly 2.5 million single nucleotide polymorphisms (SNPs) sprinkled throughout the dog genome. SNPs are variations in the DNA code, some of which contribute to diseases or the overall health of a dog. SNPs also can be used to create a set of coordinates with which to survey genetic changes, both within and across dog breeds. These efforts revealed that individual breeds have maintained a large amount of genetic variability, despite their long history of restrictive breeding. In practical terms, this means that future efforts to locate disease genes in dogs can be much narrower in scope than comparable human studies, requiring a smaller number of genetic markers and DNA samples collected from the blood or cheek from only a few hundred dogs."

Phylogenetic analysis in molecular evolutionary genetics.

2005-12-01T20:18:00.000-08:00

Entrez PubMed: "Recent developments of statistical methods in molecular phylogenetics are reviewed. It is shown that the mathematical foundations of these methods are not well established, but computer simulations and empirical data indicate that currently used methods such as neighbor joining, minimum evolution, likelihood, and parsimony methods produce reasonably good phylogenetic trees when a sufficiently large number of nucleotides or amino acids are used. However, when the rate of evolution varies extensively from branch to branch, many methods may fail to recover the true topology. Solid statistical tests for examining the accuracy of trees obtained by neighbor joining, minimum evolution, and least-squares method are available, but the methods for likelihood and parsimony trees are yet to be refined. Parsimony, likelihood, and distance methods can all be used for inferring amino acid sequences of the proteins of ancestral organisms that have become extinct."

Phylogenetic analysis in molecular evolutionary genetics. Nei M. Annu Rev Genet. 1996;30:371-403.

Parsimony, likelihood, and the role of models in molecular phylogenetics.

2005-12-01T20:15:00.000-08:00

Entrez PubMed: "Methods such as maximum parsimony (MP) are frequently criticized as being statistically unsound and not being based on any 'model.' On the other hand, advocates of MP claim that maximum likelihood (ML) has some fundamental problems. Here, we explore the connection between the different versions of MP and ML methods, particularly in light of recent theoretical results. We describe links between the two methods--for example, we describe how MP can be regarded as an ML method when there is no common mechanism between sites (such as might occur with morphological data and certain forms of molecular data). In the process, we clarify certain historical points of disagreement between proponents of the two methodologies, including a discussion of several forms of the ML optimality criterion. We also describe some additional results that shed light on how much needs to be assumed about underlying models of sequence evolution in order to successfully reconstruct evolutionary trees."

Parsimony, likelihood, and the role of models in molecular phylogenetics. Steel M, Penny D. Mol Biol Evol. 2000 Jun;17(6):839-50. Free Full Text Article

Gene family evolution and homology: genomics meets phylogenetics.

2005-12-01T20:09:00.000-08:00

Entrez PubMed: "With the advent of high-throughput DNA sequencing and whole-genome analysis, it has become clear that the coding portions of the genome are organized hierarchically in gene families and superfamilies. Because the hierarchy of genes, like that of living organisms, reflects an ancient and continuing process of gene duplication and divergence, many of the conceptual and analytical tools used in phylogenetic systematics can and should be used in comparative genomics. Phylogenetic principles and techniques for assessing homology, inferring relationships among genes, and reconstructing evolutionary events provide a powerful way to interpret the ever increasing body of sequence data. In this review, we outline the application of phylogenetic approaches to comparative genomics, beginning with the inference of phylogeny and the assessment of gene orthology and paralogy. We also show how the phylogenetic approach makes possible novel kinds of comparative analysis, including detection of domain shuffling and lateral gene transfer, reconstruction of the evolutionary diversification of gene families, tracing of evolutionary change in protein function at the amino acid level, and prediction of structure-function relationships. A marriage of the principles of phylogenetic systematics with the copious data generated by genomics promises unprecedented insights into the nature of biological organization and the historical processes that created it."

Gene family evolution and homology: genomics meets phylogenetics. Thornton JW, DeSalle R. Annu Rev Genomics Hum Genet. 2000;1:41-73.

Vestige: maximum likelihood phylogenetic footprinting.

2005-12-01T20:05:00.000-08:00

Entrez PubMed: "Phylogenetic footprinting is the identification of functional regions of DNA by their evolutionary conservation. This is achieved by comparing orthologous regions from multiple species and identifying the DNA regions that have diverged less than neutral DNA. "

Vestige: maximum likelihood phylogenetic footprinting. Wakefield MJ, Maxwell P, Huttley GA. BMC Bioinformatics. 2005 May 29;6(1):130. Free Full Text Article

An evolutionary model for protein-coding regions with conserved RNA structure.

2005-12-01T19:54:00.000-08:00

Entrez PubMed: "Here we present a model of nucleotide substitution in protein-coding regions that also encode the formation of conserved RNA structures. In such regions, apparent evolutionary context dependencies exist, both between nucleotides occupying the same codon and between nucleotides forming a base pair in the RNA structure. The overlap of these fundamental dependencies is sufficient to cause 'contagious' context dependencies which cascade across many nucleotide sites. Such large-scale dependencies challenge the use of traditional phylogenetic models in evolutionary inference because they explicitly assume evolutionary independence between short nucleotide tuples. In our model we address this by replacing context dependencies within codons by annotation-specific heterogeneity in the substitution process. Through a general procedure, we fragment the alignment into sets of short nucleotide tuples based on both the protein coding and the structural annotation. These individual tuples are assumed to evolve independently, and the different tuple sets are assigned different annotation-specific substitution models shared between their members. This allows us to build a composite model of the substitution process from components of traditional phylogenetic models. We applied this to a data set of full-genome sequences from the hepatitis C virus where five RNA structures are mapped within the coding region. This allowed us to partition the effects of selection on different structural elements and to test various hypotheses concerning the relation of these effects. Of particular interest, we found evidence of a functional role of loop and bulge regions, as these were shown to evolve according to a different and more constrained selective regime than the nonpairing regions outside the RNA structures. Other potential applications of the model include comparative RNA structure"

An evolutionary model for protein-coding regions with conserved RNA structure. Pedersen JS, Forsberg R, Meyer IM, Hein J. Mol Biol Evol. 2004 Oct;21(10):1913-22. Epub 2004 Jun 30. Free Full Text Article

Modeling mitochondrial protein evolution using structural information.

2005-12-01T19:53:00.000-08:00

Entrez PubMed: "We present two new models of protein sequence evolution based on structural properties of mitochondrial proteins. We compare these models with others currently used in phylogenetic analyses, investigating their performance over both short and long evolutionary distances. We find that our models that incorporate secondary structure information from mitochondrial proteins are statistically comparable with existing models when studying 13 mitochondrial protein data sets from eutherian mammals. However, our models give a significantly improved description of the evolutionary process when used with 12 mitochondrial proteins from a broader range of organisms including fungi, plants, protists, and bacteria. Our models may thus be of use in estimating mitochondrial protein phylogenies and for the study of processes of mitochondrial protein evolution, in particular for distantly related organisms."

Modeling mitochondrial protein evolution using structural information. Lio P, Goldman N. J Mol Evol. 2002 Apr;54(4):519-29.

Artificial Mammalian Origin Of Replication

2005-12-01T08:28:00.000-08:00

Artificial Mammalian Origin Of Replication: "Dr. Dutta and colleagues recruited known mammalian replication initiation factors (either ORC or CDC6) to a defined GAL4 DNA-binding site on a plasmid, demonstrating that replication initiation factor recruitment is sufficient to specify a DNA replication origin.
The researchers have extended the classic transcription factor reporter assay to work for any eukaryotic replication initiation factor.

The artificial mammalian replication origin will enable scientists to explore the mechanism of replication initiation, as well as 'provide a new direction for creating vectors for gene therapy that are less mutagenic than current integrating vectors and that do not require viral proteins,' explains Dr. Dutta."

Molecular Identification of Cyanobacteria Associated with Stromatolites from Distinct Geographical Locations

2005-11-27T13:16:00.000-08:00

Mary Ann Liebert, Inc. - Astrobiology - 2(3):271: "Modern stromatolites represent a significant resource for studying microbial ecology and evolution. A preliminary investigation was undertaken employing specific genetic probes to characterize the cyanobacteria responsible for stromatolite construction in a range of environments, including microbial mats found in Australia not previously examined with molecular methods. Isolates of cyanobacteria were collected from stromatolites in thermal springs, hypersaline lakes, and oceanic fringes on two continents. A polymerase chain reaction specific for DNA of cyanobacterial 16S rRNA was developed, the resulting products of the DNA amplification reaction were sequenced, and the data were used to infer relatedness between the isolates studied and other members of the cyanobacterial radiation. Complete sequence was generated for the region from position 27 to 408 for 13 strains of cyanobacteria associated with stromatolites. All stromatolite-derived sequences were most closely related to cyanobacteria, as indicated by local sequence alignment. It was possible to correlate genetic identity with morphological nomenclatures and to expand the phylogeny of benthic cyanobacteria. These inferences were also expanded to temporal variation in the dominant resident cyanobacterial species based on sampling of surface and core sinter laminations. Under the methods employed, only one cyanobacterial strain was detected in each sample, suggesting the possible dominance of a specific clonal population of cyanobacteria at any one time in the biota of the samples tested. The data indicate that internal core samples of a stromatolite at least 10 years old can be successfully analyzed by DNA-based methods to identify preserved cyanobacteria. "

Brett A. Neilan, Brendan P. Burns, David A. Relman, Donald R. Lowe Molecular Identification of Cyanobacteria Associated with Stromatolites from Distinct Geographical Locations Aug 2002, Vol. 2, No. 3: 271-280 Mary Ann Liebert, Inc. - Astrobiology - 2(3):271

DNA methyltransferases

2005-11-27T10:30:00.000-08:00

HHMI News: Biotechnology's Newest Chemical Tool: "The enzymes at the heart of the study, known as DNA methyltransferases, are one of the tools cells use to turn genes on and off. By adding a simple cluster of four atoms - a carbon atom attached to three hydrogens, known to chemists as a methyl group - to specific bases within a DNA sequence, methyltransferases can effectively shut a gene off. Methylation plays an important role in embryonic development, genomic imprinting, and carcinogenesis because it regulates gene expression."

Silenced Gene In Worm Shows Role In Regeneration

2005-11-25T07:57:00.000-08:00

Silenced Gene In Worm Shows Role In Regeneration: "Elimination of smedwi-2 not only leads to an inability to mount a regenerative response after amputation, but also to the eventual demise of unamputated animals along a reproducible series of events, that is, regression of the head tip, curling of the body and tissue disintegration. These defects are very similar to what is observed after the planarian stem cells are destroyed by lethal doses of irradiation. The key difference, however, is that the irradiation-like defects observed in animals devoid of smedwi-2 occur even though the stem cells are still present in the organism."

Normal Chromosome Ends Elicit A Limited DNA Damage Response

2005-11-24T16:16:00.000-08:00

Normal Chromosome Ends Elicit A Limited DNA Damage Response: "Verdun and his colleagues found that several well-known members of the DNA damage response machinery -- recruited by the now unprotected telomeres -- congregate at the tips of chromosomes.

'We believe that the localization of repair proteins to chromosome ends, and detection of telomeres as damage at this precise time are necessary to trigger the re-formation of a protective telomeric structure,' says Karlseder, an assistant professor in the Regulatory Biology Laboratory.

In contrast to damaged strands of DNA, they hypothesize, the repair process never gets fully underway at telomeres. Instead, the very tips of the chromosomes are looped back, tucked in and covered with telomeric proteins.

Repairing telomeres would randomly fuse whole chromosomes end-to-end. During the next cell division the sorting mechanism, which ensures that each daughter cell receives a full complement of chromosomes would inevitably rip the fused chromosomes apart.

"Such fusion breakage cycles scramble the genome over time, and cause genome instability, which is a hallmark of cancer cells," explains Karlseder. "This demonstrates the importance of telomeres in preserving genome integrity and preventing cancer development.""