Hideki Noguchi

S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex

The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.

MetaGene: prokaryotic gene finding from environmental genome shotgun sequences

Exhaustive gene identification is a fundamental goal in all metagenomics projects. However, most metagenomic sequences are unassembled anonymous fragments, and conventional gene-finding methods cannot be applied. We have developed a prokaryotic gene-finding program, MetaGene, which utilizes di-codon frequencies estimated by the GC content of a given sequence with other various measures. MetaGene can predict a whole range of prokaryotic genes based on the anonymous genomic sequences of a few hundred bases, with a sensitivity of 95% and a specificity of 90% for artificial shotgun sequences (700 bp fragments from 12 species). MetaGene has two sets of codon frequency interpolations, one for bacteria and one for archaea, and automatically selects the proper set for a given sequence using the domain classification method we propose. The domain classification works properly, correctly assigning domain information to more than 90% of the artificial shotgun sequences. Applied to the Sargasso Sea dataset, MetaGene predicted almost all of the annotated genes and a notable number of novel genes. MetaGene can be applied to wide variety of metagenomic projects and expands the utility of metagenomics.

DNA sequence and comparative analysis of chimpanzee chromosome 22

Human–chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.Human–chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.

Human chromosome 11 DNA sequence and analysis including novel gene identification

Chromosome 11, although average in size, is one of the most gene- and disease-rich chromosomes in the human genome. Initial gene annotation indicates an average gene density of 11.6 genes per megabase, including 1,524 protein-coding genes, some of which were identified using novel methods, and 765 pseudogenes. One-quarter of the protein-coding genes shows overlap with other genes. Of the 856 olfactory receptor genes in the human genome, more than 40% are located in 28 single- and multi-gene clusters along this chromosome. Out of the 171 disorders currently attributed to the chromosome, 86 remain for which the underlying molecular basis is not yet known, including several mendelian traits, cancer and susceptibility loci. The high-quality data presented here—nearly 134.5 million base pairs representing 99.8% coverage of the euchromatic sequence—provide scientists with a solid foundation for understanding the genetic basis of these disorders and other biological phenomena.

Reply to “Has the chimpanzee Y chromosome been sequenced?”

854 VOLUME 38 | NUMBER 8 | AUGUST 2006 | NATURE GENETICS includes 1.7 Mb of contiguous, non–X-degenerate sequence not examined in the earlier publication. The remaining 1.5 Mb reported by the authors is a superficial sampling of the ‘ampliconic’ portions of the chimpanzee Y chromosome. The ampliconic regions of primate Y chromosomes are of great biological and medical interest3–10. These regions are difficult but not impossible to sequence systematically and comprehensively (see refs. 3 and 5 and our unpublished results), and, in man, they comprise 10.2 Mb, or nearly half of the Y chromosome’s male-specific euchromatin3. If a similar fraction of the chimpanzee Y chromosome is ampliconic, then large and biologically significant portions of the chromosome have yet to be sequenced and analyzed. The authors1 reported more genes within the X-degenerate regions of the chimpanzee and human Y chromosomes than did investigators in earlier studies2,3, but these additions, we suggest, do not withstand scrutiny. Unlike prior studies of the human and chimpanzee Y chromosomes2,3, the authors’ inferences were based on very limited electronic analyses and were not validated experimentally. This may explain why several pseudogenes or disrupted genes—some explicitly identified as such in earlier studies—were treated as functional genes despite previous experimental evidence to the contrary (Supplementary Note and Supplementary Figure 1 online). These include the TMSB4Y and USP9Y pseudogenes on the chimpanzee Y chromosome2, the GYG2 pseudogene on the human and chimpanzee Y chromosomes2,3 and the CD24L4 pseudogene on the human Y chromosome. Finally, the authors appear to have overestimated the nucleotide divergence between the two chimpanzee Y chromosomes represented by the PTB1 and CHORI-251 libraries. We aligned the PTB1 and CHORI-251 sequences (Supplementary Tables 1–3 online; sequence alignments can be found at http://jura.wi.mit.edu/page) and found their divergence to be 0.002%, or roughly 20 times lower than the 0.0422% reported by the authors1. (The authors similarly overestimated divergence between PTB1 and a third chimpanzee Y chromosome, represented by the RPCI-43 library; our sequence alignments can be found at http://jura.wi.mit. edu/page. Note that all CHOR-251 and RPCI-43 sequences included in our alignments with PTB1 were publicly available, as finished sequence, prior to the study by Kuroki et al.) Our calculation of divergence between the Y chromosomes of PTB1 and CHORI-251 is so low (∼1 in 50,000 nucleotides) that sequencing errors (estimated at less than 1 in 200,000 nucleotides in each study) could account for about one in every three substitutions that appear to differentiate the chromosomes (Supplementary Note). It is unclear how the authors calculated a divergence 20-fold higher than ours when comparing the same sequences.