Complete genome sequence and analysis of the Streptomyces aureofaciens phage mu1/6.

The entire double-stranded DNA genome of the Streptomyces aureofaciens phage mu1/6 was sequenced and analyzed. Its size is 38.194 kbp with an overall molar G+C content of 71.2 %. Fifty-two potential open reading frames (orfs) were identified, divided into two oppositely transcribed regions. In the left arm of the mu1/6 genome, an identified putative integrase and possible regulation proteins were identified. The rightwards transcribed region contains genes organized into apparently four functional units responsible for: (i) replication, (ii) DNA packaging and head assembly, (iii) tail morphogenesis, and (iv) lysis. Putative functions were assigned to twelve orfs based on bioinformatic analysis or experimental substantiation. Comparative analysis with three complete genomes of streptomycete phages revealed resemblance with respect to the organization of their genes into functional modules. Closer relationship was observed only between mu1/6 and S. venezuelae phage VWB.

Polyunsaturated fatty acids of marine origin upregulate mitochondrial biogenesis and induce beta-oxidation in white fat.

AIMS/HYPOTHESIS: Intake of n-3 polyunsaturated fatty acids reduces adipose tissue mass, preferentially in the abdomen. The more pronounced effect of marine-derived eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on adiposity, compared with their precursor alpha-linolenic acid, may be mediated by changes in gene expression and metabolism in white fat. METHODS: The effects of EPA/DHA concentrate (6% EPA, 51% DHA) admixed to form two types of high-fat diet were studied in C57BL/6J mice. Oligonucleotide microarrays, cDNA PCR subtraction and quantitative real-time RT-PCR were used to characterise gene expression. Mitochondrial proteins were quantified using immunoblots. Fatty acid oxidation and synthesis were measured in adipose tissue fragments. RESULTS: Expression screens revealed upregulation of genes for mitochondrial proteins, predominantly in epididymal fat when EPA/DHA concentrate was admixed to a semisynthetic high-fat diet rich in alpha-linolenic acid. This was associated with a three-fold stimulation of the expression of genes encoding regulatory factors for mitochondrial biogenesis and oxidative metabolism (peroxisome proliferator-activated receptor gamma coactivator 1 alpha [Ppargc1a, also known as Pgc1alpha] and nuclear respiratory factor-1 [Nrf1] respectively). Expression of genes for carnitine palmitoyltransferase 1A and fatty acid oxidation was increased in epididymal but not subcutaneous fat. In the former depot, lipogenesis was depressed. Similar changes in adipose gene expression were detected after replacement of as little as 15% of lipids in the composite high-fat diet with EPA/DHA concentrate, while the development of obesity was reduced. The expression of Ppargc1a and Nrf1 was also stimulated by n-3 polyunsaturated fatty acids in 3T3-L1 cells. CONCLUSIONS/INTERPRETATION: The anti-adipogenic effect of EPA/DHA may involve a metabolic switch in adipocytes that includes enhancement of beta-oxidation and upregulation of mitochondrial biogenesis.

Identification of a holin encoded by the Streptomyces aureofaciens phage micro1/6; functional analysis in Escherichia coli system.

An open reading frame encoding an 88 amino acid protein was present downstream of the previously characterized endolysin of Streptomyces aureofaciens phage micro1/6. Structural analysis of its sequence revealed features characteristic for holin. This open reading frame encoding the putative holin was amplified by polymerase chain reaction and cloned into the expression vector pET-21d(+). Synthesis of the holin-like protein resulted in bacterial cell death but not lysis. The holmicro1/6 gene was able to complement the defective lambda S allele in the nonsuppressing Escherichia coli HB101 strain to produce phage progeny, This fact suggests that the proteins encoded by both phage genes have analogous function, i.e. the streptomycete holin induces nonspecific lesions in the cytoplasmic membrane, through which the lambda endolysin gains an access to its substrate, the cell wall. The concomitant expression of both S. aureofaciens holmicro 1/6 and lambda endolysin in E. coli resulted in abrupt cell lysis. This result provided further evidence that the product of holmicro 1/6 gene is a holin.

Identification and characterization of an endolysin encoded by the Streptomyces aureofaciens phage mu 1/6.

An open reading frame homologous to the genes encoding several cell-wall hydrolyzing enzymes was identified on the genome of actinophage mu 1/6. This open reading frame encoding the putative endolysin was amplified by polymerase chain reaction and cloned into the expression vector pET-21a. This gene consisted of 1182 bp encoding a 393 amino acid polypeptide with a molar mass of 42.1 kDa. The gene product was overexpressed in Escherichia coli, and then the lytic enzyme was purified by a two-step chromatographic procedure. When applied exogenously, the endolysin of phage mu 1/6 was active against all tested Streptomyces strains but did not affect other bacteria. The amino acid sequence showed a high homology with a putative amidase of the Streptomyces phase phi C31. Downstream of the endolysin gene, an open reading frame encoding an 88 amino acid protein was identified. Structural analysis of its sequence revealed features characteristics for holin.

Characterization of Amphioxus AmphiVent, an evolutionarily conserved marker for chordate ventral mesoderm.

Structure and developmental expression are described for amphioxus AmphiVent, a homolog of vertebrate Vent genes. In amphioxus, AmphiVent-expressing ventral mesoderm arises at midneurula by outgrowth from the paraxial mesoderm, but in vertebrates, Vent-expressing ventral mesoderm originates earlier, at the gastrula stage. In other embryonic tissues (nascent paraxial mesoderm, neural plate, endoderm, and tailbud), AmphiVent and its vertebrate homologs are expressed in similar spatiotemporal domains, indicating conservation of many Vent gene functions during chordate evolution. The ventral mesoderm evidently develops precociously in vertebrates because their relatively large embryos probably require an early and extensive deployment of the mesoderm-derived circulatory system. The vertebrate ventral mesoderm, in spite of its strikingly early advent, still resembles the nascent ventral mesoderm of amphioxus in expressing Vent homologs. This coincidence may indicate that Vent homologs in vertebrates and amphioxus play comparable roles in ventral mesoderm specification.

Characterization of mammalian orthologues of the Drosophila osa gene: cDNA cloning, expression, chromosomal localization, and direct physical interaction with Brahma chromatin-remodeling complex.

The osa gene of Drosophila melanogaster encodes a nuclear protein that is a component of the Brahma chromatin-remodeling complex. Osa is required for embryonic segmentation, development of the notum and wing margin, and photoreceptor differentiation. In these tissues, osa mutations have effects opposite to those caused by wingless (wg) mutations, suggesting that osa functions as an antagonist of wg signaling. Here we describe the cloning and characterization of mammalian orthologues of osa. Three evolutionarily conserved domains were identified in Osa family members: the N-terminal Bright domain and C-terminally located Osa homology domains 1 and 2. RNase protection analysis indicates a widespread expression of the Osa1 gene during mouse development, in adult tissues, and in cultured cell lines. The Osa1 gene was localized to mouse chromosome 4, within the region syntenic to chromosomal position 1p35-p36 of its human counterpart. We present evidence that the OSA1 product is localized in the nucleus and associates with human Brahma complex, which suggests evolutionarily conserved function for Osa in gene regulation between mammals and Drosophila.

The Rhodobacter capsulatus genome.

The genome of Rhodobacter capsulatus has been completely sequenced. It consists of a single chromosome containing 3.5 Mb and a circular plasmid of 134 kb. This effort, started in 1992, began with a fine-structure restriction map of an overlapping set of cosmids that covered the genome. Cosmid sequencing led to a gapped genome that was filled by primer walking on the chromosome and by using lambda clones. Methods had to be developed to handle strong stops in the high GC (68%) inserts. Annotation was done with the ERGO system at Integrated Genomics, as was the reconstruction of the cell's metabolism. It was possible to recognize 3709 orfs of which functional assignments could be made with high confidence to 2392 (65%). Unusual features include the presence of numerous cryptic phage genomes embedded in the chromosome.

The human cystathionine beta-synthase (CBS) gene: complete sequence, alternative splicing, and polymorphisms.

Cystathionine beta-synthase [CBS; l-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration and is the enzyme deficient in classical homocystinuria. In this report, we describe the molecular cloning and the complete nucleotide sequence of the human CBS gene. We report a total of 28,046 nucleotides of sequence, which, in addition to the CBS gene, contains approximately 5 kb of the 5' flanking region. The human CBS gene contains 23 exons ranging from 42 to 209 bp. The 5' UTR is formed by 1 of 5 alternatively used exons and 1 invariably present exon, while the 3' UTR is encoded by exons 16 and 17. We also describe the identification of two alternatively used promoter regions that are GC rich (approximately 80%) and contain numerous putative binding sites for Sp1, Ap1, Ap2, and c-myb, but lack the classical TATA box. The CBS locus contains an unusually high number of Alu repeats, which may predispose this gene to deleterious rearrangements. Additionally, we report on a number of DNA sequence repeats that are polymorphic in North American and European Caucasians.

Identification and tissue distribution of human cystathionine beta-synthase mRNA isoforms.

Cystathionine beta-synthase (CBS) catalyzes the irreversible, serine-dependent conversion of homocysteine to cystathionine via a transsulfuration pathway. CBS deficiency not only is the leading cause of homocystinuria, an inherited genetic disorder, but may contribute to cardiovascular disease as well. We isolated three new isoforms of human CBS mRNA from a human liver cDNA library. We designate these CBS mRNAs as CBS 3, CBS 4, and CBS 5, and the CBS mRNAs reported previously by Kraus et al. (1993) (Hum. Mol. Genet. 2, 1933-1938) and Kruger and Cox (1994) (Proc. Natl. Acad. Sci. USA 91, 6614-6618) as CBS 1 and CBS 2, respectively. Sequence analyses show that the only difference among the five CBS mRNAs is at the beginning of the 5'-untranslated region. Tissue distribution studies reveal that liver and pancreas have the highest amounts of CBS mRNAs. CBS mRNA is present in all regions of the brain tested. We also report the differential distribution of CBS mRNA isoforms in tissues, showing that pancreas contains all five CBS isoforms and the liver has four CBS mRNA isoforms, CBS 1-4. The kidney contains only CBS 1 and CBS 2. In human fetal tissues, CBS 2 is present in the liver and kidney. PCR-based quantitative analyses of CBS mRNA isoforms in human liver demonstrate that CBS 1 and CBS 2 are the major species, with CBS 2 being more abundant, while CBS 3-5 are the minor species. Furthermore, results from our human liver cDNA screening and primer extension experiments show that each of the five CBS transcripts begins with a different exon, suggesting that CBS gene transcription might be regulated by more than one promoter.

Bacteriophage B103: complete DNA sequence of its genome and relationship to other Bacillus phages.

The genome of Bacillus subtilis bacteriophage B103 consists of double-stranded linear DNA 18,630 bp long. The DNA was sequenced, and the sequence was compared with DNA sequences of closely related phages, namely the members of the phage phi29 family. Among them, phage Nf was shown to be the most closely related to B103. Comparisons of several open reading frames (ORFs) among the family members helped to identify genes 1 and 5. A cluster of ORFs between genes 16 and 17 contains two ORFs with partial homology with two phi29 ORFs located in the same region. There are three more ORFs in this region of B103 with good ribosome binding sites (RBS) and optimal codon usage that are not homologous to any of the phi29 ORFs. The function of these five ORFs remains unexplained. It was shown that major promoters characterized in phi29 are retained in B103. Where many substitutions occur in the vicinity of a promoter, at least the -10 and -35 boxes are conserved.

Sequence of a 189-kb segment of the chromosome of Rhodobacter capsulatus SB1003.

Cosmids from the 1A3-1A10 region of the complete miniset were individually subcloned by using the vector M13 mp18. Sequences of each cosmid were assembled from about 400 DNA fragments generated from the ends of these phage subclones and merged into one 189-kb contig. About 160 ORFs identified by the CodonUse program were subjected to similarity searches. The biological functions of 80 ORFs could be assigned reliably by using the WIT and Magpie genome investigation tools. Eighty percent of these recognizable ORFs were organized in functional clusters, which simplified assignment decisions and increased the strength of the predictions. A set of 26 genes for cobalamin biosynthesis, genes for polyhydroxyalkanoic acid metabolism, DNA replication and recombination, and DNA gyrase were among those identified. Most of the ORFs lacking significant similarity with reference databases also were grouped. There are two large clusters of these ORFs, one located between 45 and 67 kb of the map, and the other between 150 and 183 kb. Nine of the loosely identified ORFs (of 15) of the first of these clusters match ORFs from phages or transposons. The other cluster also has four ORFs of possible phage origin.

Sequence analysis of the bovine herpesvirus type 1 genes homologous to the DNA polymerase (UL30), the major DNA-binding protein (UL29) and ICP18.5 assembly protein (UL28) genes of herpes simplex virus.

The nucleotide sequence of a 10.5 kb region (map position 0.332 to 0.410) of bovine herpesvirus type 1 (BHV-1) was determined. This region contained three open reading frames (ORFs) homologous to herpes simplex virus DNA polymerase catalytic subunit (DNApol, UL30), major DNA-binding protein (MDBP, UL29) and ICP18.5 assembly protein (ICP18.5, UL28). The BHV-1 DNApol. MDBP and ICP18.5 ORFs were 1246, 1203 and 826 amino acids long with a calculated molecular mass of 134.2 kDa, 124.4 kDa and 86.9 kDa, respectively. They showed a high homology with alphaherpesvirus homologs despite large differences in the G + C content of the UL30-UL28 segment ranging from 44.4% for varicella zoster virus to 71.5% for BHV-1. Particularly well conserved among Alphaherpesvirinae are the putative functional domains of the DNApol and MDBP proteins which are discussed. Phylogenetic analysis revealed that BHV-1 clustered in the Varicellovirus genus with the animal D-type viruses. In this group, the BHV-1 position was shown to vary according to the investigated genes. Indeed, pseudorabies virus clustered with BHV-1 in the DNApol tree but with equine herpesvirus 1 in the ICP18.5 tree.

[A method for detection of germinal mutations in the p53 tumor suppressor gene]. / Metodika detekce zárodecných mutací v tumor supresorovém genu p53.

BACKGROUND: The tumour suppressor gene p53 is exhibits somatic mutations in a high proportion of human tumours. In addition, there are cancer families suffering from the Li-Fraumeni syndrome, the members of which carry germ line mutations in this gene. The carriers of the p53 germ line mutations have a high risk of developing tumours. The genetic diagnosis of carriership of the mutation in the tumour family members is important for preventive measures and for eventual tumour therapy modification. METHODS AND RESULTS: We have developed a method for the detection of germ line mutations in the p53 gene based on non-radioactive SSCP and direct sequencing of PCR products. We have proved the efficiency of the method by finding known mutations in eight tumour cell lines. In our collection of tumour families we have detected polymorphisms in exons 4 and 6 of the p53 gene. In one family which conformed to the criteria of the Li-Fraumem syndrome we have found a novel germ line mutation in exon 5. CONCLUSIONS: The method developed by us is very simple and sensitive. The germ line mutations in the p53 gene are very rare.

Gene contents in a 31-kb segment at the left genome end of bovine herpesvirus-1.

We report the nucleotide sequence of a 31-kb segment at the left genome end of bovine herpesvirus-1 (BHV-1) and show that it comprises 19 different open reading frames (ORFs), including seven which have been described previously (circ, dUTPase, UL49.5, alpha TIF, VP8, glycoprotein C, and ribonucleotide reductase small subunit). The new sequence resulted in a correction at the C-terminus of glycoprotein C. All 19 ORFs exhibited strong amino acid sequence homology to the gene products of other alphaherpesviruses. The BHV-1 ORFs were arranged colinearly with the prototype sequence of herpes simplex virus 1 (HSV-1) in the range of the UL54 to UL37 genes. No BHV-1 homologs of the HSV-1 UL56, UL55, and UL45 genes were identified. The BHV-1 circ gene was the only gene without a HSV-1 counterpart. The additional ORFs 1 and 2 found at the left genome end of equine herpesvirus-1 (EHV-1) were absent in BHV-1. Among the newly sequenced BHV-1 ORFs are homologs of ICP27 (UL54), glycoprotein K (UL53), helicase-primase (UL52), DNA polymerase accessory protein (UL42), ribonucleotide reductase large subunit (UL39), and several virion proteins (UL49, UL46, UL43, UL41, UL38, UL37), most of which are strongly conserved in all herpesviruses. The possible functions of the proteins encoded within the sequenced region are assessed and features found are discussed.

Sequencing and analysis of 51 kb on the right arm of chromosome XV from Saccharomyces cerevisiae reveals 30 open reading frames.

We have sequenced a region of 51 kb of the right arm from chromosome XV of Saccharomyces cerevisiae. The sequence contains 30 open reading frames (ORFs) of more than 100 amino acid residues. Thirteen new genes have been identified. Thirteen ORFs correspond to known yeast genes. One delta element and one tRNA gene were identified. Upstream of the RPO31 gene, encoding the largest subunit of RNA polymerase III, lies a Abf1p binding site. The nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDBJ nucleotide sequence databases under the Accession Number X90518.

A method for preparing M13 or pUC libraries for sequencing DNA with high G + C or A + T contents.

A simple method is described to generate M13 or pUC libraries from DNA with a very high G + C or A + T content. The G + C-rich DNA is partially digested with HinPI or HpaII restriction enzymes and cloned into the vector linearized in its multiple cloning site with AccI. The A + T-rich DNA is partially digested with TspI and cloned into the EcoRI-linearized vector. These libraries are suitable for large-scale DNA sequencing.

Sequence analysis of the UL39, UL38, and UL37 homologues of bovine herpesvirus 1 and expression studies of UL40 and UL39, the subunits of ribonucleotide reductase.

We sequenced the region of the bovine herpesvirus type 1 (BHV-1) genome corresponding to map units 0.172-0.230 (7964 bp), representing the UL39, UL38, and UL37 homologues of herpes simplex virus which encode the large subunit of ribonucleotide reductase (RR) and components of the viral capsid and the tegument, respectively. To discriminate between two potential initiator AUGs of the UL39 gene, the 5' end of the mRNA was mapped by S1 nuclease protection assays. Comparison of the amino acid sequences of the three BHV-1 proteins with analogous polypeptides from several other herpesviruses revealed significant levels of homology. We also compared the expression kinetics of the large (R1, UL39) versus the small (R2, UL40) RR subunits during the course of in vitro BHV-1 infection by Western blotting using specifically developed and calibrated antisera. Our results show that the R1 protein was synthesized earlier than its R2 counterpart. Moreover, the R1 protein accumulated to a higher level than the R2 protein even though the R2 transcript was in greater abundance than the R1 mRNA. This is discussed with regard to the translational efficiency of their transcripts.

Nucleotide sequence analysis of a 30-kb region of the bovine herpesvirus 1 genome which exhibits a colinear gene arrangement with the UL21 to UL4 genes of herpes simplex virus.

We report the nucleotide sequence of the 19-kb HindIII fragment B of bovine herpesvirus 1 (BHV-1) DNA and adjacent parts of the HindIII A and L fragments, which together span a still completely uncharted 30-kb region located between the glycoprotein H gene and the right end of the unique long segment. The analysis revealed 17 complete open reading frames (ORFs) and 2 ORFs that were interrupted by potential splice donor and acceptor sites. All of these ORFs exhibited strong amino acid sequence homology to the gene products of other alphaherpesviruses. The BHV-1 ORFs were arranged colinearly with the prototype sequence of herpes simplex virus 1 in the range of the UL21 to UL4 genes. Colinearity was also observed with the genes of betaherpesviruses and gamma herpesviruses, although not all ORFs exhibited clear sequence homology. The possible functions of the proteins encoded within the sequenced region are assessed and features found are discussed. Unexpected findings include the following: high amino acid sequence conservation among alphaherpesviruses despite large differences in G + C content, ranging from 45% for varicella zoster virus to 72% for BHV-1; high similarity with other UL20 proteins at the predicted structural level in spite of relatively low amino acid homology; and a 2-kb open reading frame overlapping UL19 in the opposite sense and exhibiting high amino acid similarity to the same area of pseudorabies virus.

Promoter, spliced leader, and coding sequence for BICP4, the largest of the immediate-early proteins of bovine herpesvirus 1.

We report the complete nucleotide sequence of the bovine herpesvirus 1 (BHV-1) immediate-early gene encoding BICP4, the homolog of the ICP4 protein of herpes simplex virus. Combined with previous mapping studies, the sequence analysis revealed that the transcript for BICP4 consisted of a noncoding leader RNA (exon 1; 0.35 kb) separated by an intron (0.46 kb) from the main body (exon 2; 4.1 kb). The open reading frame for BICP4 (1343 amino acid residues) started 27 nt after the splice site and extended across exon 2 for most of its length, BICP4 contained two domains of high homology (regions 2 and 4), which had been recognized earlier to be most conserved in the ICP4 homologs of alpha-herpesviruses and to be functionally important. These domains were flanked by three regions of lower but still discernible homology. Unique features of BICP4 were two large clusters of glutamic acid residues near the end of region 3, and the displacement of a polyserine tract to region 5, which in all other ICP4 homologs residues near the end of region 1. Transient expression assays showed that BICP4 repressed its own promoter and activated other herpes-virus genes. The 8.1-kb sequence summarized here completes analysis of the inverted repeats of the BHV-1 genome; it includes a segment (2.5 kb) upstream of the BICP4 gene apparently devoid of coding sequences but containing numerous scattered transcription signals.

Immediate-early RNA 2.9 and early RNA 2.6 of bovine herpesvirus 1 are 3' coterminal and encode a putative zinc finger transactivator protein.

Bovine herpesvirus 1 (BHV-1) contains three major immediate-early (IE) genes involved in regulation of the productive cycle of replication. Two spliced IE RNAs, IER4.2 (4.2 kb) and IER2.9 (2.9 kb), are under the control of a single promoter; IER1.7 (1.7 kb) is transcribed from a different promoter in the opposite direction. Examining the kinetics of transcription, we found that the IER4.2/2.9 promoter was turned off at the end of the IE period. An alternative promoter became active, directing synthesis of an unspliced early RNA, ER2.6 (2.6 kb), which was colinear with the second exon of IER2.9 except for its 5' end in the intron about 10 bases upstream of the splice site. Sequence analysis revealed a single open reading frame common to IER2.9 and ER2.6 with a coding potential of 676 amino acids. The putative protein, named p135, contained a cysteine-rich zinc finger domain near the N terminus with homology to ICP0 of herpes simplex virus type 1, to protein 61 of varicella-zoster virus, to early protein 0 of pseudorabies virus, and to other viral and cellular proteins. The remaining parts of p135 exhibited only limited homology, mainly with pseudorabies virus protein 0, but the entire sequence was highly conserved between two strains of BHV-1 (K22 and Jura). The latency-related antisense transcript covered a large portion of ER2.6 excluding the zinc finger coding region. In transient expression assays, p135 activated a variety of promoters, including that for ER2.6, but repressed the IER1.7 promoter. Thus, p135 combines functional characteristics of ICP0, a strong transactivator, and of protein 61, a repressor. BHV-1 seems to have evolved a subtle mechanism to ensure the continued synthesis of p135 while turning off IER4.2, which encodes p180, the herpes simplex virus type 1 ICP4 homolog.