摘要:

ABSTRACTPyrococcus furiosusis a marine microorganism with the unusual ability to grow optimally at 100°C. It is classified as a member of the domain Archaea (Archaebacteria). We present studies on the genome ofP. furiosus, consisting of a precise determination of the size of the chromosome, 2.05 mb, and an analysis of sequence data from cDNA and genomic libraries. The sequence analysis included a total of 1176 sequences, representing 14.7% of the genome, which were compared with the current databases using the BLAST X algorithm. Expressed sequence tag (EST) analysis is skewed toward the repeated detection of highly expressed genes, for example, ribosomal RNA, inP. furiosus. The relatively small size of the genome implies a high coding density, and randomly chosen genomic sequences provide good performance in gene discovery with relatively fewer identical hits compared with the EST database. The recovery of database matches with P(n) ≤ 1.0e−05was 30% of the total sequences tested. We estimate that the genome contains approximately 1800 genes, and this study has provided evidence for 309 genes. The average G+C content of the sequences obtained was 41.2%, and no repetitive sequences were detected. Genes from Eukarya and Bacteria are almost equally represented in the matches obtained in this study. The homologs representing central metabolism exhibit preferential similarity to their homologs from bacteria, despite the availability of eukaryotic homologs in the databases. Several characteristically eukaryotic protein homologs were found with functions in transcription, translation, and membrane transport. We propose that the archaeal genome is a mosaic, consisting of ancient gene sequences related to eukaryal homologs and more recently acquired genes, many of which are related to bacterial metabolic functions. Further sequencing and phylogenetic studies are needed to confirm this hypothesis, which predicts that there was extensive lateral genetic transfer among Bacteria, Eukarya, and Archaea during a period when the original gene pool was expanding into the early lineages of

ISSN:1070-2830    

DOI:10.1089/gst.1996.1.37

年代:1996

数据来源: MAL

摘要:

ABSTRACTIdentification of quantitative alterations in the gene expression during small cell lung cancer (SCLC), if sufficiently characterized, may result in novel molecular markers that may be useful in the diagnosis and treatment of human small cell lung cancer. The recently developed mRNA differential display technique has been used to identify differentially expressed sequence tags (EST), short complementary DNA fragments corresponding to mRNA that are differentially expressed in SCLC as compared with normal human bronchial epithelial cell lines as control. DNA sequencing followed by computer search against sequences present in Genbank and EMBL DNA databases indicated that one tag was novel and two had high homology with reported genes: the lissencephaly-1 gene involved in Miller-Dieker syndrome and human peptide-binding protein, which is a new member of the heat shock protein 70 (hsp70) family. Lissencephaly-1 gene maps to a region of chromosome 17p13.3, which has been found to be repeatedly deleted in SCLC. This is the first report, to our knowledge, on the tags of the genes differentially expressed between normal human bronchial epithelial and SCLC cells. Loss of their expression in SCLC could contribute to tumor formation or progression or both.

ISSN:1070-2830    

DOI:10.1089/gst.1996.1.47

年代:1996

数据来源: MAL

摘要:

ABSTRACTRecentEscherichia coligenome sequencing efforts in the 76–81.5 min and 92.8–0.1 min regions have revealed three new operons or gene clusters concerned with sugar metabolism, which we designatesga(6 ORFs),sgb(4 ORFs), and sgc (6 ORFs). All of these operons encode proteins homologous to pentose-phosphate-4-epimerases (sgaandsgb) or pentose-phosphate-3-epimerases (sgc), indicating that these operons are involved in the metabolism of pentoses or pentitols.sgaandsgc(but notsgb) encode also proteins of the bacterial phosphotransferase system (PTS), whereassgaandsgbencode three non-PTS proteins (one of which is the pentose-phosphate-4-epimerase homologue) that are homologous to each other. The two PTS protein homologues in thesgaoperon are members of (1) the family of mannitol-specific and fructose-specific IIA proteins and (2) the family of lactose-specific and cellobiose-specific IIB proteins. A IIC PTS homologue was not found in thesgaoperon, but a permease-like protein designated SgaT with 12 putative transmembrane helical segments may provide this transport function. Although thesgboperon lacks ORF homologous to PTS proteins, it contains a cryptic gene encodingL-xylulose kinase. Thesgcgene cluster encodes two PTS proteins homologous to (1) the mannitol-specific and fructose-specific IIA proteins and (2) the galactitol IIC protein. It also encodes a transcriptional regulator of the DeoR family, a pentose-phosphatase-3-epimerase homologue, and an ORF homologous to a protein encoded in the recently describedfrvoperon. Computer analyses of the DNA sequences and the encoded protein sequences are presented, and the potential roles of these operons in carbohydrate metabolism are discus

ISSN:1070-2830    

DOI:10.1089/gst.1996.1.53

年代:1996

数据来源: MAL

摘要:

ABSTRACTCurrent methods of forensic DNA identification at our facilities use PCR for typing single nucleotide and tandem repeat polymorphisms. Unfortunately, these PCR-based methods are relatively expensive and time consuming and are not well suited for total automation. The ligase detection reaction (LDR) when used in conjunction with PCR offers distinct advantages. In LDR, two adjacent primers hybridize to the target and are ligated only when there is perfect complementarity at the junction. SinceTaqDNA ligase used in LDR is thermostable, several rounds of thermal cycling may be used to unambiguously distinguish any single nucleotide polymorphisms. We have developed a coupled multiplex PCR-LDR assay to type single base variations at 12 biallelic loci, giving a power of discrimination of 1.12 × 105. The 12 loci are PCR amplified in a single reaction using a unique two-step method that produces similar amounts of multiplexed products without the need to carefully adjust primer concentrations or PCR conditions. Following PCR, these products are used in a single LDR to generate products that are resolved and typed on an Applied Biosystems 373 DNA sequencer, creating an LDR profile. Our ability to easily generate similar amounts of product in a 12-locus multiplex PCR amplification is the basis for expanding the assay to type 30 biallelic loci to give a theoretical power to discriminate one individual in 1012

ISSN:1070-2830    

DOI:10.1089/gst.1996.1.77

年代:1996

数据来源: MAL

摘要:

ABSTRACTThe expressed sequence tag (EST) strategy is a useful approach for describing gene diversity and identifying changes in expression patterns between different populations of cells. We constructed two directionally cloned cDNA libraries from human Jurkat T cell leukemia cells synchronized in either the G1or the S phase of the cell cycle. Sequence analysis of 3398 randomly selected clones from G1phase and 3428 randomly selected clones from S phase was carried out for the purpose of comparing gene expression profiles. EST sequencing identified 1728 distinct transcripts from the G1phase library and 1623 distinct transcripts from the S phase library. Approximately 13% of these transcripts appeared to be differentially expressed between the G1 and S phases of the cell cycle. Among the differentially expressed genes were alpha k-1 tubulin, alpha enolase, CDC p55, and ubiquitin. The human homologue of cyclin B2 is also differentially expressed in the G1phase vs the S phase of the cell cycle. Northern blot analysis of mRNA levels in cells from either the G1or S phase of the cell cycle correlated well with the quantitation of mRNA from EST profile analysis.

ISSN:1070-2830    

DOI:10.1089/gst.1996.1.89

年代:1996

数据来源: MAL