Nucleotide sequence analysis of DNA. IV. Complete nucleotide sequence of the left-hand cohesive end of coliphage 186 DNA.
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Adenosine Triphosphate↗
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Nucleotide sequence analysis of DNA. X. Complete nucleotide sequence of the cohesive ends of bacteriophage phi80 DNA.
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Base Sequence↗
A model for high-throughput automated DNA sequencing and analysis core facilities.
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Academies and Institutes↗
The DNA sequence and analysis of human chromosome 6.
Chromosome 6 is a metacentric chromosome that constitutes about 6% of the human genome. The finished sequence comprises 166,880,988 base pairs, representing the largest chromosome sequenced so far. The entire sequence has been subjected to high-quality manual annotation, resulting in the evidence-supported identification of 1,557 genes and 633 pseudogenes. Here we report that at least 96% of the protein-coding genes have been identified, as assessed by multi-species comparative sequence analysis, and provide evidence for the presence of further, otherwise unsupported exons/genes. Among these are genes directly implicated in cancer, schizophrenia, autoimmunity and many other diseases. Chromosome 6 harbours the largest transfer RNA gene cluster in the genome; we show that this cluster co-localizes with a region of high transcriptional activity. Within the essential immune loci of the major histocompatibility complex, we find HLA-B to be the most polymorphic gene on chromosome 6 and in the human genome.
Animals↗
The DNA sequence and analysis of human chromosome 13.
Chromosome 13 is the largest acrocentric human chromosome. It carries genes involved in cancer including the breast cancer type 2 (BRCA2) and retinoblastoma (RB1) genes, is frequently rearranged in B-cell chronic lymphocytic leukaemia, and contains the DAOA locus associated with bipolar disorder and schizophrenia. We describe completion and analysis of 95.5 megabases (Mb) of sequence from chromosome 13, which contains 633 genes and 296 pseudogenes. We estimate that more than 95.4% of the protein-coding genes of this chromosome have been identified, on the basis of comparison with other vertebrate genome sequences. Additionally, 105 putative non-coding RNA genes were found. Chromosome 13 has one of the lowest gene densities (6.5 genes per Mb) among human chromosomes, and contains a central region of 38 Mb where the gene density drops to only 3.1 genes per Mb.
Chromosome Mapping↗
DNA sequence and analysis of human chromosome 9.
Chromosome 9 is highly structurally polymorphic. It contains the largest autosomal block of heterochromatin, which is heteromorphic in 6-8% of humans, whereas pericentric inversions occur in more than 1% of the population. The finished euchromatic sequence of chromosome 9 comprises 109,044,351 base pairs and represents >99.6% of the region. Analysis of the sequence reveals many intra- and interchromosomal duplications, including segmental duplications adjacent to both the centromere and the large heterochromatic block. We have annotated 1,149 genes, including genes implicated in male-to-female sex reversal, cancer and neurodegenerative disease, and 426 pseudogenes. The chromosome contains the largest interferon gene cluster in the human genome. There is also a region of exceptionally high gene and G + C content including genes paralogous to those in the major histocompatibility complex. We have also detected recently duplicated genes that exhibit different rates of sequence divergence, presumably reflecting natural selection.
Base Composition↗
Novel truncating mutations in the ClC-5 chloride channel gene in patients with Dent's disease.
BACKGROUND: Dent's disease is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets and eventual renal failure. The disease is caused by mutations in the X-linked chloride channel CLCN5 gene, which encodes a 746-amino-acid protein expressed in renal tubules. These mutations have been reported in unrelated families from the UK, USA, Japan and other countries. We were interested in identifying additional mutations in the CLCN5 coding region of Spanish patients with Dent's disease. METHODS: Five patients from three unrelated Spanish families were studied. Leukocyte genomic DNA from patients and their relatives was used with CLCN5-specific primers for polymerase chain reaction amplification of the coding region and exon-intron boundaries. Amplified products were analysed by single-strand conformational polymorphism analysis, DNA sequencing and restriction enzyme analysis. RESULTS: Low-molecular-weight proteinuria and hypercalciuria were detected in all the patients, nephrocalcinosis in two patients, and rickets or osteopenia in three patients. We identified three new CLCN5 mutations consisting of two nonsense mutations, Leu433Stop and Arg718Stop, and an insertional frameshift mutation, 65insT, which results in a stop at codon 98. These three mutations predict truncated ClC-5 proteins that, respectively, lack 314, 649 and 28 amino acids at the carboxy terminus, and are likely to result in loss of function. These mutations were shown to co-segregate with the disease in each of the three families. CONCLUSIONS: Our study is the first to characterize mutations in the CLCN5 gene in Spanish patients with Dent's disease and expands the spectrum of CLCN5 mutations associated with this disease.
Adult↗
Cloning and sequence analysis of a Marek's disease virus origin binding protein (OBP) reveals strict conservation of structural motifs among OBPs of divergent alphaherpesviruses.
Marek's disease virus (MDV) is a highly cell-associated avian herpesvirus. In its natural host, MDV induces Marek's disease (MD), a lethal condition characterized by malignant lymphoma of T cells. Although symptoms of MD may be prevented by vaccination, no practical pharmacological method of control has been widely accepted. Viral replication represents a point at which pharmacological control of herpesvirus infection may be most successful. However, this requires detailed knowledge of viral replication proteins. Studies in HSV-1 DNA replication implicate the UL9 protein as a key initiator of replication. For example, binding of UL9 to HSV-1 origins is a prerequisite for assembly of additional replication proteins. In this study, a protein, whose apparent molecular size is similar to that of HSV-1 UL9, was identified in extracts of MDV infected cells by western blot analysis with anti-HSV-1 UL9 antibody. A putative MDV UL9 gene was subsequently identified through sequencing of MDV genome fragments (BamHI G and C). Extended DNA sequence analysis revealed an open reading frame (ORF) which could encode a protein homologous to HSV-1 UL9. The MDV UL9 ORF encodes 841 amino acids, producing a sequence 49% identical to HSV-1 UL9 and 46% identical to VZV gene 51 product (VZV UL9). MDV UL9 shares numerous structural motifs with HSV-1 and VZV UL9 proteins, including six conserved N-terminal helicase motifs, an N-terminal leucine zipper motif, a C-terminal pseudo-leucine zipper sequence, and a putative helix-turn-helix structure.
Alphaherpesvirinae↗
Sequencing by hybridization (SBH): advantages, achievements, and opportunities.
Efficient DNA sequencing of the genomes of individual species and organisms is a critical task for the advancement of biological sciences, medicine and agriculture. Advances in modern sequencing methods are needed to meet the challenge of sequencing such megabase to gigabase quantities of DNA. Two possible strategies for DNA sequencing exist: direct methods, in which each base position in the DNA chain is determined individually (e.g., gel sequencing or pyrosequencing), and indirect methods, in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the DNA chain. One promising indirect method is sequencing by hybridization (SBH), in which sets of oligonucleotides are hybridized under conditions that allow detection of complementary sequences in the target nucleic acid. The unprecedented sequence search parallelism of the SBH method has allowed development of high-throughput, low-cost, miniaturized sequencing processes on arrays of DNA samples or probes. Newly developed SBH methods use DNA ligation to combine relatively small sets of short probes to score potentially tens of millions of longer oligonucleotide sequences in a target DNA. Such combinatorial approaches allow analysis of DNA samples of up to several kilobases (several times longer than allowed by current direct methods) for a variety of DNA sequence analysis applications, including de novo sequencing, resequencing, mutation/SNP discovery and genotyping, and expression monitoring. Future advances in biochemistry and implementation of detection methods that allow single-molecule sensitivity may provide the necessary miniaturization, specificity, and multiplexing efficiency to allow routine whole genome analysis in a single solution-based hybridization experiment.
Base Sequence↗
Alterations of the p53 tumor suppressor gene in carcinoma in situ of the testis.
BACKGROUND: Carcinoma in situ (CIS) is regarded as the precursor of all histologic variants of testicular germ cell tumors except spermatocytic seminoma. For a variety of human malignancies, alterations of the p53 tumor suppressor gene have been identified as prognostic factors for a poor clinical course. Discussions of the occurrence of p53 gene alterations in testicular carcinoma have been controversial. Immunohistochemical detection of the p53 oncoprotein has been reported in four of eight CIS cell areas adjacent to mature teratoma. The majority of investigations have failed to demonstrate p53 gene alterations on the DNA level in testicular carcinoma specimens. However, the genetic analysis of testicular carcinoma is complicated by the histologic variety of tumors, resulting in a mixture of subtypes undergoing moleculargenetic analysis. In the present study, CIS cells identified in normal testicular tissue adjacent to different testicular tumors were examined for alterations of the p53 tumor suppressor gene. The authors believed that the detection of p53 alterations in CIS of the testis would not only support the idea of malignant potential in CIS but might also demonstrate the involvement of the p53 tumor suppressor gene in the development of germ cell cancers. METHODS: CIS cells were identified in the normal testicular tissue adjacent to 18 seminomatous and nonseminomatous germ cell tumors by histopathologic criteria and the immunohistochemical staining reaction for placental-like alkaline phos phase, a highly specific marker for testicular CIS. About 20-50 CIS cells per tumor were collected by a microdissection technique and were subjected to RNA. SSCP analysis and additional DNA-sequence analysis. RESULTS: In 12 of 18 cases (66%), RNA-SSCP analysis of the microdissected CIS cells revealed mutational band shifting at the p53 gene locus. In 7 of 18 cases (39%). the results of SSCP analysis were confirmed by DNA sequencing. DNA-sequence analysis revealed missense point mutations in the p53 gene in four cases (exon 5, codons 158, 170, and 176; exon 6, codon 213) and silent mutations in two cases (exon 5, codon 178; exon 6, codon 213). In one case, the identical missense point mutation (exon 5, codon 176) was detected in the CIS cells and also in the associated germ cell tumor. In two of six cases, different mutations were found in the CIS cells and the testicular tumor. In three cases in which DNA sequencing revealed point mutations in CIS cells, alterations of the p53 gene could not be detected in the examination of the associated tumor specimen. CONCLUSIONS: These findings appear to support the concept that malignant biologic characteristics are present in the in situ stage of testicular germ cell tumors. This is the first report demonstrating monoclonal development of a manifested testicular carcinoma from associated CIS cells on the basis of a certain mutational event in the p53 gene sequence. The involvement of p53 gene alterations in precursor cells of testicular carcinoma appears likely.
Alkaline Phosphatase↗
Localization and sequence analysis of chloroplast DNA sequences of Chlamydomonas reinhardii that promote autonomous replication in yeast.
Four distinct chloroplast DNA segments from Chlamydomonas reinhardii of 400, 415, 730 and 2300 bp which promote autonomous replication in yeast have been mapped on the chloroplast genome. Plasmids carrying these chloroplast DNA fragments are unstable in yeast when the cells are grown under non-selective conditions. Sequence analysis of three of these chloroplast ARS regions (autonomously replicating sequences in yeast) reveals a high AT content, numerous short direct and inverted repeats and the presence of at least one element in each region that is related to the yeast ARS consensus sequence. A/T TTTATPuTTT A/T. These three chloroplast regions share, in addition, two common elements of 10 and 11 bp which may play a role in promoting autonomous replication.
Journal Article↗
Well-differentiated villoglandular adenocarcinoma of the uterine cervix: oncogene/tumor suppressor gene alterations and human papillomavirus genotyping.
Twelve well-differentiated villoglandular adenocarcinomas (WDVAs) of the uterine cervix were retrospectively analyzed for the presence and specific genotype of human papillomavirus (HPV), tumor suppressor loss (p53, MCC, APC, BRCA1), cancer gene mutation (K-ras-2, exons 1 and 2, p53 exons 5 to 8), and oncogene amplification (c-erbB-2/HER-2/neu, int-2). Tissue for genetic evaluation was obtained by microdissection, using 4-micron-thick histology sections of archival, formalin-fixed, paraffin-embedded specimens. Genotyping involved nucleic acid amplification and DNA sequencing with gene-specific oligonucleotides and L1 region consensus primers for common strains of HPV. Point mutation and HPV strain determination were accomplished by DNA sequence analysis. Tumor suppressor gene loss and oncogene amplification were performed by allelic imbalance analysis in informative subjects based on DNA sequence and microsatellite-length polymorphisms. HPV was present in all tumors and consisted of type 16 (n = 5, 42%) and type 18 (n = 7, 58%) strains, which have been closely associated with cervical neoplasia. K-ras-2 and p53 genes did not manifest point mutational damage. There was no evidence of oncogene amplification or tumor suppressor gene loss. The presence of HPV in all 12 tumors supports the role of HPV infection in the molecular pathogenesis of this uncommon neoplasm. The absence of associated oncogene or tumor suppressor gene damage is consistent with indolent biological behavior and the favorable prognosis of this unusual tumor.
Adenocarcinoma↗
The Magnaporthe grisea DNA fingerprinting probe MGR586 contains the 3' end of an inverted repeat transposon.
The Magnaporthe grisea repeat (MGR) sequence MGR586 has been widely used for population studies of the rice blast fungus, and has enabled classification of the fungal population into hundreds of genetic lineages. While studying the distribution of MGR586 sequences in strains of M. grisea, we discovered that the plasmid probe pCB586 contains a significant amount of single-copy DNA. To define precisely the boundary of the repetitive DNA in pCB586, this plasmid and four cosmid clones containing MGR586 were sequenced. Only 740 bp of one end of the 2.6-bp insert in the pCB586 plasmid was common to all clones. DNA sequence analysis of cosmid DNA revealed that all the cosmids contained common sequences beyond the cloning site in pCB586, indicating that the repetitive DNA in the fingerprinting clone is part of a larger element. The entire repetitive element was sequenced and found to resemble an inverted repeat transposon. This putative transposon is 1.86 kb in length and has perfect terminal repeats of 42 bp, which themselves contain direct repeats of 16 bp. The internal region of the transposon possesses one open reading frame which shows similarity at the peptide level to the Pot2 transposon from M. grisea and Fot1 from Fusarium oxysporum. Hybridization studies using the entire element as a probe revealed that some strains of M. grisea, whose DNA hybridized to the pCB586 probe, entirely lacked MGR586 transposon sequences.
Amino Acid Sequence↗
Establishing the rDNA IGS structure of Cannabis sativa.
The rDNA intergenic spacer (IGS) structure of Cannabis sativa was established and can be used for classification and identification of this species. In this study, DNA fragments of rDNA IGS were amplified by PCR from Cannabis sativa plant extracts and a 1387 bp fragment was obtained. DNA sequence analysis revealed six different repeat motifs. In the middle of the IGS sequence, there were three sequence motifs, and the same three sections of DNA were then repeated with minor variation in sequence. The terminal region of the IGS was composed of another three different repeat units; multiple copies of these terminal repeat motifs were present in no discernible order. Within six repeat motifs, point variations were observed in five. The DNA sequence of the locus was compared with all the plant sequences registered in GenBank by the Fasta program of GCG software with the result that this DNA fragment was significantly different from any other DNA sequence recorded to date. The most similar sequence was that of Hops (Humulus lupulus), but with a similarity of only 88.9% over 579 bp. These specific and complex variations of IGS may be related to the species and geographic distributions.
Base Sequence↗
Molecular analysis of the gene encoding a novel chitin-binding protease from Alteromonas sp. strain O-7 and its role in the chitinolytic system.
Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to alpha-chitin and beta-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35 degrees C, respectively, and even at 10 degrees C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.
Alteromonas↗
Identification of the Agrobacterium tumefaciens C58 T-DNA genes e and f and their impact on crown gall tumour formation.
DNA sequence analysis of the 4.4 kilobases (kb) Eco RI fragment 14 from T-DNA of Agrobacterium tumefaciens C58 revealed three open reading frames. One of them (945 bp) was supposed to encode the transcript e, the function of which has not been identified to date. Furthermore, a so far undescribed open reading frame (1035 bp) was identified, located in the centre of the Eco RI fragment 14 and termed gene f. The third open reading frame encoded the carboxy-terminal part of the agrocinopine synthase (Acs). The gene e-encoded protein showed significant homologies to the gene products of the Agrobacterium rhizogenes rolB gene and the Agrobacterium tumefaciens gene 5. Both gene products are supposed to regulate the plant's reaction on auxin. Depending on the plant species tested, Agrobacterium strains carrying mutations in gene e induced only small or almost no detectable crown gall tumours. According to these mutational studies and the protein homologies observed, the gene e product is suggested to be involved in tumour formation. Infection of several plant species with Agrobacterium carrying a mutated gene f, as well as expression of the gene f in transgenic tobacco plants did not lead to visible morphological changes. Therefore, in contrast to gene e, the gene f seems not to be essential for tumour formation. In order to study whether gene f is an active gene, its expression in agrobacteria and plants was monitored by translational lacZ fusion. In planta, the putative gene f-promoter mediates a tissue-specific expression pattern. Although gene f was expressed in free-living agrobacteria as well as in transgenic plants, the function of the f locus remained unclear. DNA homology studies with the f gene region revealed a mosaic-like DNA structure, indicating that this locus might be the result of genetic exchanges between different Agrobacterium strains during evolution.
Agrobacterium tumefaciens↗
The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences.
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Internet↗
Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism.
Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.
Amino Acid Sequence↗