Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. I. Sequence features in the 1 Mb region from map positions 64% to 92% of the genome

The contiguous sequence of 1,003,450 bp spanning map positions 64% to 92% of the genome of Synechocystis sp. strain PCC6803 has been deduced. Computer analysis of the sequence predicts that this region contains at least 818 potential ORFs, in which 255 (31%) were either genes that had already been identified or their homologues, 84 (10%) were homologues to registered hypothetical genes, and 149 (18%) showed weak similarities to reported genes. The remaining 330 ORFs showed no apparent similarity to any reported genes or carried no significant protein motifs. The potential ORFs as a whole occupied 86% of the sequenced region, implying compact arrangement of genes in the genome. As to the structural RNA genes, one rRNA operon consisting of 5,028 bp and at least 11 species of tRNA genes were identified. It is noteworthy that 10 out of the 11 tRNA species showed significant sequence similarities to tRNAs reported in plant chloroplasts. As other notable unique sequences, three classes of IS-like elements each with characteristics typical of IS elements were identified, and a typical unit of WD(Trp-Asp)-repeats which have only been detected in the regulatory proteins of eukaryotes was identified within the large 5,079-bp ORF located at map position 69%.     

Analysis of 94 kb of the chlorella virus PBCV-1 330-kb genome: map positions 88 to 182

Analysis of 94 kb of DNA, located between map positions 88 and 182 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 195 open reading frames (ORFs) 65 codons or longer. One hundred and five of the 195 ORFs were considered major ORFs. Twenty-six of the 105 major ORFs resembled genes in the databases including three chitinases, a chitosanase, three serine/threonine protein kinases, two additional protein kinases, a tyrosine protein phosphatase, two ankyrins, an ornithine decarboxylase, a copper/zinc-superoxide dismutase, a proliferating cell nuclear antigen, a DNA polymerase, a fibronectin-binding protein, the yeast Ski2 protein, an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease, and an amidase. The genes for the 105 major ORFs were evenly distributed along the genome and, except for one noncoding 1788-nucleotide stretch, the genes were close together. Unexpectedly, a 900-bp region in the 1788-bp noncoding sequence resembled a CpG island.     

The physical map of the human RET proto-oncogene

The RET proto-oncogene, a transmembrane tyrosine kinase receptor, is involved in the development of at least five different disease phenotypes. RET is activated through somatic rearrangements in a number of cases of papillary thyroid carcinoma while germ-line point mutations are associated with three inherited cancer syndromes MEN 2A, MEN 2B and FMTC. Moreover, point mutations or heterozygous deletions of RET are found in the dominant form of Hirschsprung disease or congenital colonic aganglionosis. We cloned the entire RET genomic sequence in a contig of cosmids encompassing 150 kb, from the CA repeat sTCL-2 to the region upstream the RET promoter, and established the position of the 20 exons of the RET gene with respect to a detailed restriction map based on eight endonucleases. A new highly polymorphic CA repeat sequence was identified within intron 5 of RET (RET-INT5). Finally the orientation of RET on chromosome 10q11.2 made it possible to orientate three other genes rearranged with RET in papillary thyroid carcinomas, namely H4/D10S170 on 10q21, R1 alpha on 17q23 and RFG2/Ele1 on 10q11.2.     

Two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins

A two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins has been established. About 250 spots were detected after silver staining and polypeptides from 24 spots have been N-terminally sequenced. Major proteins already described in bull seminal plasma, like PDC-109 and aSFP, have been located on the map; proteins not yet reported in male reproductive tracts have been evidenced; for some polypeptides showing a previously unknown N-terminal sequence, structural similarities with proteins described in other organisms have been found. A reference map of seminal plasma proteins could be useful in relating protein pattern changes to physiopathological events influencing the reproductive sphere.     

Construction of a contiguous 874-kb sequence of the Escherichia coli -K12 genome corresponding to 50.0-68.8 min on the linkage map and analysis of its sequence features

The contiguous 874.423 base pair sequence corresponding to the 50.0-68.8 min region on the genetic map of the Escherichia coli K-12 (W3110) was constructed by the determination of DNA sequences in the 50.0-57.9 min region (360 kb) and two large (100 kb in all) and five short gaps in the 57.9-68.8 min region whose sequences had been registered in the DNA databases. We analyzed its sequence features and found that this region contained at least 894 potential open reading frames (ORFs), of which 346 (38.7%) were previously reported, 158 (17.7%) were homologous to other known genes, 232 (26.0%) were identical or similar to hypothetical genes registered in databases, and the remaining 158 (17.7%) showed no significant similarity to any other genes. A homology search of the ORFs also identified several new gene clusters. Those include two clusters of fimbrial genes, a gene cluster of three genes encoding homologues of the human long chain fatty acid degradation enzyme complex in the mitochondrial membrane, a cluster of at least nine genes involved in the utilization of ethanolamine, a cluster of the secondary set of 11 hyc genes participating in the formate hydrogenlyase reaction and a cluster of five genes coding for the homologues of degradation enzymes for aromatic hydrocarbons in Pseudomonas putida. We also noted a variety of novel genes, including two ORFs, which were homologous to the putative genes encoding xanthine dehydrogenase in the fly and a protein responsible for axonal guidance and outgrowth of the rat, mouse and nematode. An isoleucine tRNA gene, designated ileY, was also newly identified at 60.0 min.     

A physical map of chromosome 7 of Candida albicans

As part of the ongoing Candida albicans Genome Project, we have constructed a complete sequence-tagged site contig map of chromosome 7, using a library of 3840 clones made in fosmids to promote the stability of repeated DNA. The map was constructed by hybridizing markers to the library, to a blot of the electrophoretic karyotype, and to a blot of the pulsed-field separation of the SfiI restriction fragments of the genome. The map includes 149 fosmids and was constructed using 79 markers, of which 34 were shown to be genes via determination of function or comparison of the DNA sequence to the public databases. Twenty-five of these genes were identified for the first time. The absolute position of several markers was determined using random breakage mapping. Each of the homologues of chromosome 7 is approximately 1 Mb long; the two differ by about 20 kb. Each contains two major repeat sequences, oriented so that they form an inverted repeat separated by 370 kb of unique DNA. The repeated sequence CARE2/Rel2 is a subtelomeric repeat on chromosome 7 and possibly on the other chromosomes as well. Genes located on chromosome 7 in Candida are found on 12 different chromosomes in Saccharomyces cerevisiae.     

A 37.5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes, a TCP-1-related gene, an open reading frame similar to the DAL80 gene, and a tRNA(Arg)

The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromosome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding gamma-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Cc eta subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family. Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNA(Arg3) (AGC) gene.     

Integrated map of the Schizosaccharomyces pombe genome

A restriction map of the entire Schizosaccharomyces pombe genome was constructed using two restriction enzymes (BamHI and PstI) that recognize 6 bp. The restriction map contains 420 minimally overlapping clones (miniset) and has 22 gaps. We located 126 genes, marker fragments of DNA (NotI and SfiI linking clones), and 36 transposable elements by hybridization to unique restriction fragments.     

The effect of parathyroid hormone and other metabolic factors on extrarenal acid buffering

Two regions from the genome of the virulent Lactobacillus delbrueckii subsp. lactic bacteriophage LL-H were sequenced (2330 and 12939 bp; 44% of the 34.6-kb genome). Together with the previously sequenced region containing the major capsid protein-encoding gene (2498 bp), the sequence had 21 open reading frames (ORFs) on the main coding strand. Only two putative ORFs were detected on the complementary strand. The ORFs covered 93.2% of the sequence. All but four of the ORFs were preceded by a ribosome-binding site. Only four longer non-coding stretches of sequences (175-278 nucleotides (nt) in size) were present. The longest of the non-coding regions contained an A + T-rich sequence that is surrounded by eight perfect copies of an 8-nt sequence that is present both as direct and inverted repeats. This region could represent the origin of replication. All the previously mapped structural protein-encoding genes of phage LL-H were included in the sequence. Genes were identified for the following five proteins: gp19 (encoded by gene g17), gp58 (g71), gp61 (g57), gp75 (g70) and gp89 (g88). N-terminal amino-acid sequencing was performed on gp19 and gp75, and it was found that the N-terminal Met had been post-translationally removed from both proteins.     

131I dose-dependent thyroid autoimmune disorders in children living around Chernobyl

The nucleotide sequence of 35,400 bp at approximately 10 kb from the right telomere of chromosome VII was determined. The segment contains the MAL1 locus, one of the five unlinked loci sufficient for maltose utilization. Until now, each of these loci was considered to contain three genes (for regulator, permease and alpha-glucosidase), but a fourth gene, presumably an extra alpha-glucosidase gene, was found at MAL1 adjacent to the usual cluster of three genes. The two glucosidase genes are present in opposite orientation, forming an inverted repeat structure. In addition to the four genes at MAL1, there are 11 complete, non-overlapping open reading frames (ORFs) longer than 300 bp in the sequence presented here. A new ABC transporter gene (YGR281w), required for oligomycin resistance was found (YOR1; Katzman et al., 1995), and the previously sequenced BGL2 (YGR282c), ZUO1 (YGR285c) and BIO2 (YGR286c) genes were located. The sequence of BIO2, a biotin synthetase gene, required substantial correction and the size of Bio2p is 375, rather than 356, amino acids. Two ORFs show rather weak similarities to animal genes: YGR278w to an unknown ORF of Caenorhabditis elegans and YGR284c to the murine Surf-4, a member of a cluster of at least four housekeeping genes. The remaining five ORFs do not encode known functions, but three of these show weak to high similarities to other ORFs in the Saccharomyces cerevisiae genome and one (YGR280c) codes for a particularly lysine-rich protein.     

Physical organization of subgroup B human adenovirus genomes

Cleavage sites of nine bacterial restriction endonucleases were mapped in the DNA of adenovirus type 3 (Ad3) and Ad7, representative serotypes of the "weakly oncogenic" subgroup B human adenoviruses. Of 94 sites mapped, 82 were common to both serotypes, in accord with the high overall sequence homology of DNA among members of the same subgroups. Of the sites in Ad3 and Ad7 DNA, fewer than 20% corresponded to mapped restriction sites in the DNA of Ad2 or Ad5. The latter serotypes represent the "nononcogenic" subgroup C, having only 10 to 20% overall sequence homology with the DNA of subgroup B adenoviruses. Hybridization mapping of viral mRNA from Ad7-infected cells resulted in a complex physical map that was nearly identical to the map of early and late gene clusters in Ad2 DNA. Thus the DNA sequences of human adenoviruses of subgroups B and C have significantly diverged in the course of viral evolution, but the complex organization of the adenovirus genome has been rigidly conserved.     

A genomic region of lactococcal temperate bacteriophage TP901-1 encoding major virion proteins

Two major structural proteins, MHP (major head protein) and MTP (major tail protein), from the lactococcal temperate phage TP901-1 were sequenced at their amino acid termini, and derived degenerate oligonucleotides were used to locate the corresponding genes in the phage genome. This genomic region was sequenced. The sequence characterized includes a total of 11 open reading frames (ORFs) showing an operon structure. Upstream of each ORF, except ORF b2 and ORF x, potential ribosome-binding sites were found, suggesting independent translation. However, coupled translation is suggested for ORF x and as a possibility for ORF b3 and ORF c2, which have ribosome-binding sites located more distant from their start codons. ORF b2 may be translationally fused with mhp at a low frequency. The mhp and mtp genes are transcribed as a 3.7-kb mRNA with at least six additional ORFs. The organization of the genomic region analyzed resembles that of other distantly related phages, providing possible roles for the uncharacterized ORFs.     

Nuclear medicine approaches for detection of axillary lymph node metastases

Rhopalosiphum padi virus (RhPV) is an aphid virus that has been considered a member of the Picornaviridae based on physicochemical properties. The 10,011-nt polyadenylated RNA genome of RhPV was completely sequenced. Analysis of the sequence revealed the presence of two open reading frames (ORFs). The predicted amino acid sequence of ORF1, representing the first 6600 nt of the RhPV genome, showed significant similarity to the nonstructural proteins of several plant and animal RNA viruses. Direct sequence analysis of the RhPV capsid proteins showed that ORF2, which represents the last 2900 nt, encodes the three structural proteins (28, 29, and 30 kDa). The predicted amino acid sequence of ORF2 is very similar to the corresponding regions of Drosophila C virus, Plautia stali intestine virus, and to a partial sequence from the 3' end of the cricket paralysis virus genome. The site of initiation of protein synthesis for ORF2 could not be determined from the amino acid and nucleotide sequences. ORF1 is preceded by 579 nt of noncoding RNA and the two ORFs are separated by more than 500 nt of noncoding RNA. Like picornaviruses, these regions may function to facilitate the cap-independent initiation of translation of the two ORFs. These data suggest that RhPV, Drosophila C virus, Plautia stali intestine virus, and probably cricket paralysis virus are members of a unique group of small RNA viruses that infect primarily insects.