Sequence analysis of the Streptococcus mutans fructosyltransferase gene and flanking regions.

The nucleotide sequence of the ftf gene from Streptococcus mutants GS-5 was determined. The deduced amino acid sequence indicates that the unprocessed fructosyltransferase gene product has a molecular weight of 87,600. A typical streptococcal signal sequence is present at the amino terminus of the protein. The processed enzyme is relatively hydrophilic and has a pI of 5.66. An inverted repeat structure was detected upstream from the ftf gene and may function in the regulation of fructosyltransferase expression. Sequencing of the regions flanking the gene revealed the presence of four other putative open reading frames (ORFs). Two of these, ORFs 2 and 3, appear to code for low-molecular-weight proteins containing amino acid sequences sharing homology with several gram-positive bacterial DNA-binding proteins. In addition, ORF 3 is transcribed from the ftf DNA coding strand. Partial sequencing of ORF 4 suggests that its gene product may be an extracellular protein.     

Structural analysis of plasmid pLQ510 from Moraxella catarrhalis E22.

The complete nucleotide sequence of plasmid pLQ510 from Moraxella catarrhalis strain E22 has been determined. This plasmid contained 12,082 bp with 38% GC content. Five open reading frames that encoded predicted proteins with homology to plasmid-encoded proteins from other bacteria were identified. A putative origin of replication that contained an AT-rich region followed by four direct repeats and an inverted repeat was identified.     

Sequence analysis of the 4.7-kb BamHI-EcoRI fragment of the equine herpesvirus type-1 short unique region.

To localize gene that may encode immunogens potentially important for recombinant vaccine design, we have analysed a region of the equine herpesvirus type-1 (EHV-1) genome where a glycoprotein-encoding gene had previously been mapped. The 4707-bp BamHI-EcoRI fragment from the short unique region of the EHV-1 genome was sequenced. This sequence contains three entire open reading frames (ORFs), and portions of two more. ORF1 codes for 161 amino acids (aa), and represents the C terminus of a possible membrane-bound protein. ORF2 (424 aa) and ORF3 (550 aa) are potential glycoprotein-encoding genes; the predicted aa sequences contain possible signal sequences, N-linked glycosylation sites and transmembrane domains; they also show homology to the glycoproteins gI and gE of herpes simplex virus type-1 (HSV-1), and the related proteins of pseudorabies virus and varicella-zoster virus. The predicted aa sequence of ORF4 shares no homology with other known herpesvirus proteins, but the nucleotide sequence shows a high level of homology with the corresponding region of the EHV-4 genome. ORF5 may be related to US9 of HSV-1.     

Developmental rearrangement of cyanobacterial nif genes: nucleotide sequence, open reading frames, and cytochrome P-450 homology of the Anabaena sp. strain PCC 7120 nifD element.

An 11-kbp DNA element of unknown function interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. In developing heterocysts the nifD element excises from the chromosome via site-specific recombination between short repeat sequences that flank the element. The nucleotide sequence of the nifH-proximal half of the element was determined to elucidate the genetic potential of the element. Four open reading frames with the same relative orientation as the nifD element-encoded xisA gene were identified in the sequenced region. Each of the open reading frames was preceded by a reasonable ribosome-binding site and had biased codon utilization preferences consistent with low levels of expression. Open reading frame 3 was highly homologous with three cytochrome P-450 omega-hydroxylase proteins and showed regional homology to functionally significant domains common to the cytochrome P-450 superfamily. The sequence encoding open reading frame 2 was the most highly conserved portion of the sequenced region based on heterologous hybridization experiments with three genera of heterocystous cyanobacteria.     

Characterization of a member of the highly repeated long interspersed rat DNA family with long open reading frames

A highly repetitive long interspersed sequence from rat DNA has been isolated and partly characterized. This sequence comprises at least a 1300 base-pair and a 2400 base-pair EcoRI fragment and probably additional elements. The 2400 base-pair segment has been analyzed in detail. It appears to be part of the chromosomal DNA in rat cells. The 2400 base-pair repeat is likely to be distributed over several regions in the rat genome. The 2400 base-pair segment has been cloned, mapped for restriction sites, and part of its nucleotide sequence has been determined. The 2400 base-pair sequence is a member of a typical highly repetitive long interspersed sequence with high copy number and restriction site polymorphism. There are sequence homologies to mouse and human DNA. A striking homology has been detected to the flanking sequences of a repetitive mouse DNA sequence that has been described to be located adjacent to one of the kappa-immunoglobulin variable genes. Elements in the 2400 base-pair rat repeat are transcribed in cells from most rat organs and from several continuous rat cell lines. This RNA from rat cell lines was found polyadenylated or not polyadenylated. The nucleotide sequence of parts of the 2400 base-pair DNA segment revealed open reading frames for polypeptide sequences. Such open reading frames have been detected in two different segments of the 2400 base-pair DNA repeat. Open reading frames exist in the two complementary strands in the same DNA segment. The hypothetical polypeptide whose sequence has been determined in toto has a length of 190 amino acid residues and is enriched in hydrophobic amino acids, reminiscent of the amino acid composition in membrane proteins. Hence, it is conceivable that the 2400 base-pair repeat sequence from rat DNA, at least in part, encodes messenger RNAs that might be translated into functional proteins.     

The complete nucleotide sequence of PEBV RNA2 reveals the presence of a novel open reading frame and provides insights into the structure of tobraviral subgenomic promoters.

The 3374 nucleotide sequence of RNA2 from the British PEBV strain SP5 has been determined. The RNA includes three open reading frames flanked by 5' and 3' noncoding regions of 509 and 480 nucleotides. The open reading frames specify coat protein, a 29.6K product homologous to the 29.1K product of TRV(TCM) RNA2 and a 23K product not homologous to any previously described protein. The homology demonstrated between the coat proteins of PRV, TRV and PEBV indicates a common evolutionary origin for these proteins. Upstream of each ORF are located sequences homologous to those with which subgenomic RNAs of other tobraviruses start. Subgenomic RNAs for the expression of the three ORFs may start at these points. On all five tobraviral RNA2 molecules sequenced to date, these sequences were found upstream of the coat protein ORF in association with a strongly-conserved potential secondary structural element. Similar potential structures were identified upstream of other tobraviral ORFs. These structures may contribute to the activity of the tobraviral subgenomic promoter.     

Nucleotide sequence of an infectious molecularly cloned genome of ground squirrel hepatitis virus.

We have determined the complete nucleotide sequence of an infectious cloned genome of ground squirrel hepatitis virus (GSHV), a nonpathogenic member of the hepadnavirus group. The genome is 3,311 base pairs long and contains the major open reading frames described for the related human and woodchuck hepatitis B viruses (HBV and WHV, respectively). These reading frames include genes for the major structural proteins (the surface and core antigens), unassigned open reading frames (A and B), the longer of which is presumed to encode the viral DNA polymerase, and an open reading frame preceding and continuous with the surface antigen gene. The arrangement of these open reading frames is similar to that encountered in the genomes of HBV and WHV: all of the reading frames are encoded on the same strand, they are positioned in the same fashion with respect to each other, and a large portion (at least 51%) of the genome can be translated in two reading frames. Comparisons of the predicted translational products of the three mammalian hepadnaviruses reveal 78% amino acid homology between the proteins of GSHV and WHV and 43% homology between those of GSHV and HBV. In addition, a perfect direct repeat of 10 to 11 base pairs, separated by ca. 46 to 223 base pairs, is present in the three mammalian viruses and in duck hepatitis B virus; the position of the repeats near the 5' termini of the two strands of virion DNA suggests a role in viral replication.     

Identification and characterization of an Epstein-Barr virus early antigen that is encoded by the NotI repeats.

The Epstein-Barr virus (EBV) genome is characterized by two regions carrying partially homologous clusters of short tandem repeats (NotI and PstI repeats) flanked by 1,044 and 1,045 base pairs with almost perfect homology (DL and DR, left and right duplications, respectively). Both repetitive regions are transcribed into poly(A)+ mRNA after induction of the productive EBV cycle with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and contain open reading frames. To identify the potential protein encoded by the NotI repeat open reading frame (BHLF1), two repeat units of EBV strain M-ABA were expressed using the tryptophan-regulated Escherichia coli expression vector pATH11. Rabbit antisera generated against the resulting fusion protein reacted specifically with a protein varying in molecular size between 70,000 and 90,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, found after 12-O-tetradecanoyl-phorbol-13-acetate or n-butyrate induction in various cell lines harboring EBV. In immunofluorescence tests with the BHLF1-specific antiserum, an immunofluorescence with EA-D specificity could be observed. In addition, the BHLF1 protein is exhibiting polyanion-binding activity with a maximum for single-stranded DNA. Furthermore, the fusion protein is recognized by a number of human EBV-positive sera.     

Sequence organization of repetitive elements in the flanking regions of the chloroplast ribosomal unit of Chlamydomonas reinhardii.

The flanking regions and the end of the chloroplast ribosomal unit of Chlamydomonas reinhardii have been sequenced. The upstream region of the ribosomal unit contains three open reading frames coding for 111, 117 and 124 amino acids, respectively. The latter polypeptide is partially related to the ribosomal protein L16 of E. coli. Two of the open reading frames overlap each other and are oriented in opposite direction. The region between these open reading frames and the 5' end of the 16S rRNA gene contains numerous short direct and inverted repeats which can be folded into large stem-loop structures. Sequence elements that resemble prokaryotic promoters are found in the same region. Several of the repeated elements are distributed throughout the non-coding regions of the chloroplast inverted repeat. Sequence comparison between the 5S rRNA and its gene does not reveal any significant sequence heterogeneity between the chloroplast 5S rRNA genes.     

Nucleotide sequence of the promoter-distal region of the tra operon of plasmid R100, including traI (DNA helicase I) and traD genes

The nucleotide sequence of the promoter-distal region of the tra operon of R100 was determined. There are five open reading frames in the region between traT and finO, and their protein products were identified. Nucleotide sequences of plasmid F corresponding to the junction regions among the open reading frames seen in R100 were also determined. Comparison of these nucleotide sequences revealed strong homology in the regions containing traD, traI and an open reading frame (named orfD). The TraD protein (83,899 Da) contains three hydrophobic regions, of which two are located near the amino-terminal region. This protein also contains a possible ATP-binding consensus sequence at the amino-terminal region and a characteristic repeated peptide sequence (Gln-Gln-Pro)10 at the carboxy-terminal region. The TraI protein (191,679 Da) contains the sequence motif conserved in an ATP-dependent DNA helicase superfamily in its carboxy-terminal region. The protein product of orfD, which is probably a new tra gene (named traX), contains 65% hydrophobic amino acids, especially rich in alanine and leucine. There exist non-homologous regions between R100 and F that could be represented as four I-D (insertion or deletion) loops in heteroduplex molecules. Assignment of each loop to the strand of R100 or F was , however, found to be the reverse from that previously assumed. The three I-D loops that were located between traT and traD, between traD and traI, and between traI and finO had no terminal inverted repeat sequences nor had they any homology with known insertion sequences, while the fourth was IS3, located within the finO gene of F. The sequences in the I-D loops, except IS3, may also code for proteins that are, however, likely to be nonessential for transfer of plasmids.     

DNA sequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species.

DNA hybridization experiments indicate that the genome of a tumorigenic poxvirus. Shope fibroma virus (SFV), possesses sequence homology with DNA isolated from uninfected rabbit cells. Southern blotting experiments, either with high-complexity rabbit DNA as probe and SFV restriction fragments as targets or with high-specific activity, 32P-labeled, cloned SFV sequences as probes and rabbit DNA as target, indicate that the homologous sequences map at two locations within the viral genome, one in each copy of the terminal inverted repeat sequences. Unexpectedly, Southern blots revealed that the homologous host sequences reside in a rabbit extrachromosomal DNA element. This autonomous low-molecular-weight DNA species could be specifically amplified by cycloheximide treatment and was shown by isopycnic centrifugation in cesium chloride-ethidium bromide to consist predominantly of covalently closed circular DNA molecules. DNA sequencing of pSIC-9, a cloned 1.9-kilobase fragment of the rabbit plasmid species, indicated extensive homology at the nucleotide level over a 1.5-kilobase stretch of the viral terminal inverted repeat. Analysis of open reading frames in both the plasmid and SFV DNA revealed that (i) the N-terminal 157-amino acid sequence of a potential 514-amino acid SFV polypeptide is identical to the N-terminal 157 amino acids of one pSIC-9 open reading frame, and (ii) a second long pSIC-9 open reading frame of 361 amino acids, although significantly diverged from the comparable nucleotide sequence in the virus, possessed considerable homology to a family of cellular protease inhibitors, including alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. The potential role of such cellular plasmid-like DNA species as a mediator in the exchange of genetic information between the host cell and a cytoplasmically replicating poxvirus is discussed.     

fhuC and fhuD genes for iron (III)-ferrichrome transport into Escherichia coli K-12.

The nucleotide sequence for a 1,900-base-pair region of the Escherichia coli chromosome that includes the genes fhuC and fhuD was determined. Within this sequence are two open reading frames: nucleotides 127 to 921 and nucleotides 924 to 1811. These coding regions specify a FhuC protein with an Mr of 28,423 and a mature FhuD protein with an Mr of 29,610. The deduced amino acid sequence of FhuC shows extensive homology with those of components of some bacterial transport systems which are peripheral proteins of the cytoplasmic membrane. Because the FhuD protein contains a typical signal sequence of 30 amino acids at the amino terminus and displays characteristics of a soluble protein, it may be exported into the periplasm.     

Nucleotide sequence and genomic organization of Aleutian mink disease parvovirus (ADV): sequence comparisons between a nonpathogenic and a pathogenic strain of ADV.

A DNA sequence of 4,592 nucleotides (nt) was derived for the nonpathogenic ADV-G strain of Aleutian mink disease parvovirus (ADV). The 3'(left) end of the virion strand contained a 117-nt palindrome that could assume a Y-shaped configuration similar to, but less stable than, that of other parvoviruses. The sequence obtained for the 5' end was incomplete and did not contain the 5' (right) hairpin structure but ended just after a 25-nt A + T-rich direct repeat. Features of ADV genomic organization are (i) major left (622 amino acids) and right (702 amino acids) open reading frames (ORFs) in different translational frames of the plus-sense strand, (ii) two short mid-ORFs, (iii) eight potential promoter motifs (TATA boxes), including ones at 3 and 36 map units, and (iv) six potential polyadenylation sites, including three clustered near the termination of the right ORF. Although the overall homology to other parvoviruses is less than 50%, there are short conserved amino acid regions in both major ORFs. However, two regions in the right ORF allegedly conserved among the parvoviruses were not present in ADV. At the DNA level, ADV-G is 97.5% related to the pathogenic ADV-Utah 1. A total of 22 amino acid changes were found in the right ORF; changes were found in both hydrophilic and hydrophobic regions and generally did not affect the theoretical hydropathy. However, there is a short heterogeneous region at 64 to 65 map units in which 8 out of 11 residues have diverged; this hypervariable segment may be analogous to short amino acid regions in other parvoviruses that determine host range and pathogenicity. These findings suggested that this region may harbor some of the determinants responsible for the differences in pathogenicity of ADV-G and ADV-Utah 1.     

Nucleotide sequence of soybean chloroplast DNA regions which contain the psb A and trn H genes and cover the ends of the large single copy region and one end of the inverted repeats.

The soybean chloroplast psb A gene (photosystem II thylakoid membrane protein of Mr 32 000, lysine-free) and the trn H gene (tRNAHisGUG), which both map in the large single copy region adjacent to one of the inverted repeat structures (IR1), have been sequenced including flanking regions. The psb A gene shows in its structural part 92% sequence homology with the corresponding genes of spinach and N. debneyi and contains also an open reading frame for 353 aminoacids. The aminoacid sequence of a potential primary translation product (calculated Mr, 38 904, no lysine) diverges from that of spinach and N. debneyi in only two positions in the C-terminal part. The trn H gene has the same polarity as the psb A gene and the coding region is located at the very end of the large single copy region. The deduced sequence of the soybean chloroplast tRNAHisGUG is identical with that of Zea mays chloroplasts. Both ends of the large single copy region were sequenced including a small segment of the adjacent IR1 and IR2.