Aspergillus fumigatus is the most ubiquitous opportunistic filamentous fungal pathogen of human. As an initial step toward sequencing the entire genome of A. fumigatus, which is estimated to be approximately 30 Mb in size, we have sequenced a 922 kb region, contained within 16 overlapping bacterial artificial chromosome (BAC) clones. Fifty-four percent of the DNA is predicted to be coding with 341 putative protein coding genes. Functional classification of the proteins showed the presence of a higher proportion of enzymes and membrane transporters when compared to those of Saccharomyces cerevisiae. In addition to the nitrate assimilation gene cluster, the quinate utilisation gene cluster is also present on this 922 kb genomic sequence. We observed large scale synteny between A. fumigatus and Aspergillus nidulans by comparing this sequence to the A. nidulans genetic map of linkage group VIII.
A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.
The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284, 156 base pairs. Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D. radiodurans to survive under conditions of starvation, oxidative stress, and high amounts of DNA damage were identified. Deinococcus radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell.
The complete genome sequence of Treponema pallidum was determined and shown to be 1,138,006 base pairs containing 1041 predicted coding sequences (open reading frames). Systems for DNA replication, transcription, translation, and repair are intact, but catabolic and biosynthetic activities are minimized. The number of identifiable transporters is small, and no phosphoenolpyruvate:phosphotransferase carbohydrate transporters were found. Potential virulence factors include a family of 12 potential membrane proteins and several putative hemolysins. Comparison of the T. pallidum genome sequence with that of another pathogenic spirochete, Borrelia burgdorferi, the agent of Lyme disease, identified unique and common genes and substantiates the considerable diversity observed among pathogenic spirochetes.
Chromosome 2 of Plasmodium falciparum was sequenced; this sequence contains 947,103 base pairs and encodes 210 predicted genes. In comparison with the Saccharomyces cerevisiae genome, chromosome 2 has a lower gene density, introns are more frequent, and proteins are markedly enriched in nonglobular domains. A family of surface proteins, rifins, that may play a role in antigenic variation was identified. The complete sequencing of chromosome 2 has shown that sequencing of the A+T-rich P. falciparum genome is technically feasible.
The genome of the bacterium Borrelia burgdorferi B31, the aetiologic agent of Lyme disease, contains a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs. The chromosome contains 853 genes encoding a basic set of proteins for DNA replication, transcription, translation, solute transport and energy metabolism, but, like Mycoplasma genitalium, it contains no genes for cellular biosynthetic reactions. Because B. burgdorferi and M. genitalium are distantly related eubacteria, we suggest that their limited metabolic capacities reflect convergent evolution by gene loss from more metabolically competent progenitors. Of 430 genes on 11 plasmids, most have no known biological function; 39% of plasmid genes are paralogues that form 47 gene families. The biological significance of the multiple plasmid-encoded genes is not clear, although they may be involved in antigenic variation or immune evasion.
        
Title: Interruption of myogenesis by transforming growth factor beta 1 or EGTA inhibits expression and activity of the myogenic-associated (2'-5') oligoadenylate synthetase and PKR Salzberg S, Mandelboim M, Zalcberg M, Shainberg A, Mandelbaum M Ref: Experimental Cell Research, 219:223, 1995 : PubMed
Interferon-induced proteins have been previously implicated in the regulation of cell growth. In an attempt to provide evidence for the involvement of these proteins in differentiation, the effect of transforming growth factor beta 1 (TGF-beta) and EGTA on the expression and activity of (2'-5') oligoadenylate synthetase (2-5A synthetase) and double-stranded RNA activated protein kinase (PKR) during myogenesis of rat primary skeletal muscle cultures or the myogenic cell line L8 was studied. Both TGF-beta and EGTA inhibited the fusion of myoblasts and reduced significantly the level of the muscle-specific proteins, acetylcholine receptors, and creatine kinase activity in rat primary muscle cultures. Likewise, TGF-beta exhibited a similar inhibitory effect on the fusion of L8 cells and the level of creatine kinase activity in these cells. The kinetics of 2-5A synthetase activity in both types of cells during differentiation was then established. In both types, a transient increase in activity was observed followed by a decrease thereafter. However, while the peak activity in primary muscle cultures appeared after 24 h in culture, it was observed only on the third day in L8 cells grown in differentiation medium (DM). Treatment of primary cultures with either TGF-beta or EGTA reduced the amount of 1.7-kb 2-5A synthetase-specific RNA transcripts and decreased significantly the level of 2-5A synthetase activity compared to that in untreated cultures. Western blot analysis of 2-5A synthetase proteins in untreated primary muscle cultures showed that the major species synthesized in these cells was the 43-kDa isoform of the enzyme. However, the 71-kDa isoform was clearly visible after 72 h in culture. Both TGF-beta and EGTA abrogated the appearance of all forms of 2-5A synthetase. Similarly, in L8 cells grown in DM, TGF-beta down-regulated the expression of 2-5A synthetase and reduced the level of enzymatic activity. Western blot analysis revealed the presence of the 71-kDa isoform as the major species of 2-5A synthetase in L8 cells; however, the 43-kDa isoform was also visible on the third day in DM. TGF-beta treatment resulted in a reduced amount of 2-5A synthetase proteins. The kinetics of PKR activity in L8 cells grown in DM was similar to that observed with 2-5A synthetase. Furthermore, TGF-beta strongly reduced the level of PKR activity in differentiating L8 cells.
        
Title: Infection with Moloney murine sarcoma virus inhibits myogenesis and alters the myogenic-associated (2'-5')oligoadenylate synthetase expression and activity Birnbaum M, Shainberg A, Salzberg S Ref: Virology, 194:865, 1993 : PubMed
Infection of rat skeletal muscle cultures on the first or second day in vitro with Moloney murine sarcoma virus (MSV) led to the arrest of myotube formation and to inhibition of both the synthesis of the muscle-specific proteins acetylcholine receptors and creatine kinase and the expression of the myosin light chain-2. Mos-specific RNA transcripts were readily detected at 1 day after infection indicating that viral genes were expressed in infected cells. In parallel, the expression of the cell growth-associated gene--c-myc--in uninfected muscle cultures was drastically reduced with time, while in MSV-infected myoblasts, the amount of c-myc-specific RNA transcripts gradually increased with time after infection. Under these conditions we could demonstrate that the interferon-induced gene (2'-5')oligoadenylate synthetase (2-5A synthetase) was transiently activated in uninfected muscle culture reaching a peak activity on the third day. Infection of myoblasts with murine leukemia virus did not alter the pattern of 2-5 synthetase activity observed in uninfected cells. However, infection with MSV on the second day led to a slight reduction in activity followed by a significant increase on the sixth and seventh day. Similarly, 2-5A synthetase gene expression was down-regulated with time in culture in uninfected myoblasts while re-expressed between the fourth and seventh days in MSV-infected cultures.
Differentiation of skeletal muscle involves withdrawal of myoblasts from cell replication, fusion to form multinucleated myotubes, coordinate appearance of a variety of muscle-specific proteins and the disappearance of a set of other proteins responsible for cell growth. The possible activation of the interferon (IFN) system in this process was studied. Thus, the activity of two IFN-induced enzymes known to be part of the system-(2'-5') oligoadenylate synthetase (2-5A synthetase) and double-stranded RNA-activated protein kinase as well as the expression of 2-5A synthetase coding genes were examined during myogenesis. It is demonstrated that the activity of the enzymes is transiently increased in cultured myoblasts, reaching a peak activity on the 3rd day in culture and then declining to a basal level. This peak activity precedes both cell fusion and the appearance of muscle-specific proteins--acetylcholine receptors (AChR) and creatine kinase. The same kinetics of 2-5A synthetase activity was evident in myoblasts from chick, rat or mouse origin. The enzymatic product appears to be primarily the trimer form of 2-5A, rather than a set of oligomers observed in enzymatic reactions performed on IFN-treated cells, including muscle cultures. The kinetics of 2-5A synthetase gene expression revealed that the largest amount of specific RNA transcripts appeared on the 1st day after seeding, followed by a reduction thereafter. In addition, a decrease was also observed in expression of c-myc, a cell-growth-associated protooncogene. However, an increase towards the 2nd day of both AChR and myosin light chain gene expression was evident, indicating selective regulation of gene expression during myogenesis.