MicrobiologyBytes

An epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence. Such changes are mediated by chemical modifications to chromatin on both DNA and DNA-associated histones. Post-translational covalent modifications to the flexible NH2 terminus (tail) of histones include methylation, acetylation, phosphorylation and ubiquitylation, and these are associated with the structural organization of chromatin and its transcriptional status. However, not all histone modifications are truly epigenetic, as very few satisfy the heritable part of the definition. To establish and mediate epigenetic memory, such modifications must be transmitted during DNA replication. Methylation of cytosine in CpG dinucleotides (often referred to as DNA methylation) also contributes to the epigenetic status of a gene locus. When this occurs in a CpG island adjacent to a transcription initiation site, it is generally associated with repression or silencing of transcription. Histone modification, DNA methylation and the resulting reorganisation of chromatin are closely interlinked enzyme-driven processes that determine the transcriptional status of genes, gene clusters and noncoding RNAs such as micro (mi)RNAs. Most of the epigenetic markers mentioned above are associated with transcriptional repression. Multiple additional covalent modifications to histones exist in parallel to these, resulting in a complex and context-influenced ‘histone code’ that dictates transcriptional state.

One of the key questions in the study of mammalian gene regulation is how epigenetic methylation patterns on histones and DNA are initiated and established. These stable, heritable, covalent modifications are largely associated with the repression or silencing of gene transcription, and when deregulated can be involved in the development of human diseases such as cancer. This article reviews examples of viruses and bacteria known or thought to induce epigenetic changes in host cells, and how this might contribute to disease. The heritable nature of these processes in gene regulation suggests that they could play important roles in chronic diseases associated with microbial persistence; they might also explain so-called ‘hit-and-run’ phenomena in infectious disease pathogenesis.

Epigenetic reprogramming of host genes in viral and microbial pathogenesis. Trends Microbiol. Aug 17 2010

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Transcription, the process of converting DNA into RNA (which in turn is translated into proteins by ribosomes) is carried out by the multisubunit RNA polymerase (RNAP) enzyme. Transcription is fundamental to all organisms across the three kingdoms of life – Eukarya, Bacteria, and Archaea – and can be divided into three major steps: initiation, transcription/elongation, and termination. Eukaryotes have three different nuclear RNAPs, whereas Archaea and Bacteria have one. Archaeal transcription is similar to that of eukaryotes, but initiation requires only two accessory proteins bound to DNA: transcription factor B (TFB) and TATA-box binding protein (TBP). It is believed that studies of the archaeal enzyme may shed light on the more complex eukaryotic RNAP. New research has shown that organisms which live in boiling acid read their DNA using enzymes surprisingly similar to our own, providing insight into the way in which the information stored in DNA is unlocked. This new work has shown that the enzyme that converts DNA into RNA is conserved between simple single-celled microorganism Sulfolobus and more complicated “higher” organisms, including human beings, despite a staggering 2 billion year evolutionary gulf. A new paper explores how evolution has shaped our own RNAP enzyme to accomplish more complex functions.

Many Archaea are extremophiles; living in high salt, acid or temperature environments. Transcription, the process of reading DNA to make RNA (which is in turn translated into proteins by ribosomes) is a fundamental process common to all organisms, and is carried out by the enzyme multisubunit RNA polymerase (RNAP). Eukaryotes have three different RNAPs, whereas Archaea and Bacteria have one. Archaea can serve as a wonderful model system because their simpler RNA polymerase machinery is related to the more complex eukaryotic RNAP. To start transcription, the archaeal enzyme requires two accessory proteins whilst the eukaryotic counterpart needs at least two more. This increased complexity prompts two important questions: how did our polymerase evolve from the ancestral enzyme; and how does Archaea bypass the requirement of further co-factor proteins?

New work investigates the polymerase from the Archaeon Sulfolobus shibatae using X-ray crystallography. This reveals the enzyme’s architecture which confirms its close evolutionary relationship with the eukaryotic RNAP. The research also identified a subunit novel to Sulfolobus which has no equivalent in the eukaryotic enzyme. The striking structural similarities suggest that the ancestral eukaryote used the same enzyme as the Archaeon, and that modern eukaryotic RNAP evolved by the addition of bolt-on proteins that regulate eukaryotic-specific processes. From the location and topology of the newly identified, Archaeon-only subunit, the scientists have suggested a mechanism by which Archaea do without the additional cofactors required by eukaryotes for initiating transcription. The scientists also noted that the complete structure of the archaeal polymerase illustrates how the ancestral core enzyme was modulated by addition of novel subunits, an evolutionary process that has facilitated the complexity that we see today in Eukarya.

Evolution of complex RNA polymerases: The complete archaeal RNA polymerase structure. 2009 PLoS Biol 7(5): e1000102
The archaeal RNA polymerase (RNAP) shares structural similarities with eukaryotic RNAP II but requires a reduced subset of general transcription factors for promoter-dependent initiation. To deepen our knowledge of cellular transcription, we have determined the structure of the 13-subunit DNA-directed RNAP from Sulfolobus shibatae at 3.35 A° resolution. The structure contains the full complement of subunits, including RpoG/Rpb8 and the equivalent of the clamp-head and jaw domains of the eukaryotic Rpb1. Furthermore, we have identified subunit Rpo13, an RNAP component in the order Sulfolobales, which contains a helix-turn-helix motif that interacts with the RpoH/Rpb5 and RpoA9/Rpb1 subunits. Its location and topology suggest a role in the formation of the transcription bubble.

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• Unique cell division machinery in the Archaea

A new DNA test for Human Papillomavirus (HPV) which causes cervical cancer is so much better than current methods that some gynecologists hope it will eventually replace the Pap smear in wealthy countries and other tests in poor ones. Not only could the new test for HPV save lives, scientists say that women over 30 could drop annual Pap smears and instead have the DNA test just once every 3, 5 or even 10 years, depending on which expert is asked. This optimism is based on an eight-year study of 130,000 women in India financed by the Bill and Melinda Gates Foundation and published recently in the New England Journal of Medicine:

HPV Screening for Cervical Cancer in Rural India. 2009 New Engl J. Med. 360: 1385-1394
In October 1999, we began to measure the effect of a single round of screening by testing for human papillomavirus (HPV), cytologic testing, or visual inspection of the cervix with acetic acid (VIA) on the incidence of cervical cancer and the associated rates of death in the Osmanabad district in India. In this cluster-randomized trial, 52 clusters of villages, with a total of 131,746 healthy women between the ages of 30 and 59 years, were randomly assigned to four groups of 13 clusters each. The groups were randomly assigned to undergo screening by HPV testing (34,126 women), cytologic testing (32,058), or VIA (34,074) or to receive standard care (31,488, control group). Women who had positive results on screening underwent colposcopy and directed biopsies, and those with cervical precancerous lesions or cancer received appropriate treatment. In the HPV-testing group, cervical cancer was diagnosed in 127 subjects (of whom 39 had stage II or higher), as compared with 118 subjects (of whom 82 had advanced disease) in the control group (hazard ratio for the detection of advanced cancer in the HPV-testing group, 0.47; 95% confidence interval [CI], 0.32 to 0.69). There were 34 deaths from cancer in the HPV-testing group, as compared with 64 in the control group (hazard ratio, 0.52; 95% CI, 0.33 to 0.83). No significant reductions in the numbers of advanced cancers or deaths were observed in the cytologic-testing group or in the VIA group, as compared with the control group. Mild adverse events were reported in 0.1% of screened women. In a low-resource setting, a single round of HPV testing was associated with a significant reduction in the numbers of advanced cervical cancers and deaths from cervical cancer.

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• From India to the World – A Better Way to Prevent Cervical Cancer. 2009 New Engl J. Med. 360: 1453-1455
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Human disease attributable to variola virus (VARV), the etiologic agent of smallpox, has been reported in human populations for more than 2,000 years. VARV is unique among orthopoxviruses in that it is an exclusively human pathogen. Because it has a large, slowly evolving DNA genome, researchers were able to construct a phylogeny of VARV by analyzing single nucleotide polymorphisms (SNPs) from genome sequences of 47 VARV isolates with broad geographic distributions. The results reveal two primary VARV clades, which are likely to have diverged from an ancestral African rodent-borne variola-like virus either 16,000 or 68,000 years before present (YBP), depending on which historical records (East Asian or African) are used to calibrate the molecular clock. One primary clade was represented by the Asian VARV major strains, the more clinically severe form of smallpox, which spread from Asia either 400 or 1,600 YBP. The other primary clade included both alastrim minor, a phenotypically mild smallpox described from the Americas, and isolates from West Africa. This clade diverged from an ancestral VARV either 1,400 or 6,300 YBP.
Observations of smallpox-typical skin rashes on Egyptian mummies dating from 1100 to 1580 B.C. gave credibility to theories that ancient Egypt was an early (and perhaps the earliest) smallpox endemic region. However, smallpox researchers noted that “The most striking thing about smallpox is its absence from the books of the Old and New Testaments, and also from the literature of the Greeks and Romans. Such a serious disease as variola major is very unlikely to have escaped a description by Hippocrates if it existed.” Historical records from Asia describe evidence of smallpox-like disease in medical writings from ancient China (1122 B.C.) and India (as early as 1500 B.C.). The earliest unmistakable description of smallpox first appears in the 4th century A.D. in China, the 7th century A.D. in India and the Mediterranean, and the 10th century A.D. in southwestern Asia. These early Asian descriptions could indicate that pandemic smallpox originated in East Asia. Sequence analysis indicates that divergence between VARV and rodent poxviruses occurred from 16,000 YBP to 68,000 YBP, and that VARV seems to have evolved from a pathogen of African rodents and subsequently spread out of Africa.
On the origin of smallpox: Correlating variola phylogenics with historical smallpox records
PNAS USA 2007 104:15787-15792

What does this all mean?

• In spite of concerns about bioterrorism, smallpox is no longer a major human pathogen, but understanding the origin of this disease, which has been of major importance for most of human history, offers glimpses into how we might rapidly understand new emerging diseases as they appear.
• For a long time it has been generally believed the the most probable origin for smallpox virus was in Asia, but as with yellow fever and HIV, this new research seems to show that smallpox originally came out of Africa.

Update: The end of the world? Dr Franken-Venter? Nope

Craig Venter has built a synthetic genome out of laboratory chemicals and is poised to announce the creation of the first artificial life form. A team of 20 scientists led by Nobel laureate Hamilton Smith has constructed a synthetic chromosome which is 381 genes long and contains 580,000 base pairs of DNA. The nucleotide sequence is based on the bacterium Mycoplasma genitalium which the team pared down to the bare essentials needed to support life, removing a fifth of its genetic make-up. The wholly synthetically reconstructed chromosome, which the team have called Mycoplasma laboratorium, has been tagged with watermarks for easy recognition and transplanted into a living bacterial cell to become a new life form. Venter has further heightened the controversy surrounding his potential breakthrough by applying for a patent for the synthetic bacterium.

Good idea, or not?