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PubMed2005

Human, rhesus macaque, and feline sequences highly similar to mouse mammary tumor virus sequences.

Sequences highly similar (>95%) to the mouse mammary tumor virus (MMTV) env gene have been amplified from human DNA samples, including DNA samples from patients with breast cancer (BC) and persons who did not have BC. The sequences from human DNA were distinct from the MMTV sequences used as controls in these PCR reactions, indicating that these results are not simply due to contamination. In addition to both, mouse and human-related sequences were also amplified from some monkey and cat genomic DNA samples. These products were shown to be distinct from, but highly related to, the MMTV env gene, whereas, testing of other sources (lambda phage, snake, cockroach, sea urchin, chicken, or dog) demonstrated no specific amplification. A sequence 90% similar to the MMTV group antigen gene (gag) was amplified from cat DNA. These results indicate that DNA from vertebrate species other than rodents, including some but not all humans, monkeys, and cats, can contain sequences closely related to MMTV.

Animals↗
PubMedDate not supplied

Bacterial plasmid pBR322 sequences serve as upstream activating sequences in Saccharomyces cerevisiae.

The expression of acid phosphatase (APase) from PHO5 and MF alpha-PHO5 hybrid genes is regulated by inorganic phosphate and mating type locus respectively, as well as the PHO4 and MAT alpha 1 gene products respectively. When PHO5 and MF alpha-PHO5 hybrid genes were cloned in the BamHI site of the pBR322 sequence of the yeast shuttle vectors (YRp7 or YEp9T), in one orientation they were regulated normally but in the other orientation their expression was not regulated but expressed constitutively. The pBR322 sequences present upstream of the inserted genes are responsible for the constitutive expression. By replacing the PHO5 upstream activating sequences (UAS) element with pBR322 fragments, we have identified three pBR322 sequences, from nucleotides 376 to 650, 2068 to 2116 and 2136 to 2247, which were able to promote expression of APase. A comparison of these three pBR322 fragments revealed 5' ATCGCGCGAG 3' and 5' CGGTGATGNCGG 3' to be the common sequences likely to act as UASs in Saccharomyces cerevisiae. By using synthetic oligonucleotides, it was found that both sequences are required for maximum expression of APase activity.

Acid Phosphatase↗
PubMedDate not supplied

Nucleotide sequence of the cytochrome oxidase subunit 2 and val-tRNA genes and surrounding sequences from Kluyveromyces lactis K8 mitochondrial DNA.

The nucleotide sequence of the cytochrome oxidase subunit 2 (cox2) and val-tRNA genes and surrounding regions from Kluyveromyces lactis mitochondrial DNA is reported. Analysis of the coding region shows that the codons CUN (Thr), CGN (Arg) and AUA (Met) are absent in this gene. A single sequence, ATATAAGTAA, identical to the baker's yeast mtRNA polymerase recognition site, was detected upstream of val-tRNA. This sequence is absent from regions between val-tRNA-cox2 and cox2-cox1. In addition a sequence AATAATATTCTT, identical to the mRNA processing site in other yeast mitochondrial genomes is present 32-43 bp downstream to the TAA stop codon for the cox2 gene. Another short conserved sequence of 5 bp, TCTAA, is present upstream of the coding regions of cox2 genes in several yeasts, including K. lactis, but is not present upstream of other genes. Comparison of cox2 sequences from other organisms indicates that the mitochondrial DNA of K. lactis is closely related to that of Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1995

Frequency and polymorphism of simple sequence repeats in a contiguous 685-kb DNA sequence containing the human T-cell receptor beta-chain gene complex.

The human T-cell receptor beta-chain (TCRB) gene complex spans 575 kb in chromosome region 7q35 and has been the subject of a large-scale DNA sequencing effort. A contiguous 685-kb DNA sequence from this region was searched by computer analysis for the occurrence of simple sequence repeats (microsatellites) with core sequence lengths of 2-5 nucleotides. Twenty-nine such microsatellites of repeat number n > or = 9 were found, with the majority being dinucleotide repeats. By PCR analysis, 19 were found to be polymorphic in repeat number, thus averaging one per 36 kb. These polymorphic di-, tri-, and tetranucleotide repeats had between 3 and 15 differently sized alleles each. The potential usefulness of these TCRB microsatellites for detecting disease susceptibility alleles was examined by measuring the linkage disequilibrium between these markers and flanking biallelic mutations. All but 4 microsatellites (79%) demonstrated significant linkage disequilibrium (P < 0.0001). This present study highlights the utility and potential outcomes of large-scale DNA sequencing for the identification of polymorphic simple sequence repeats.

Alleles↗
PubMed1993

Canine distemper terminal and intergenic non-protein coding nucleotide sequences: completion of the entire CDV genome sequence.

Sequences critical for the transcription and replication functions of canine distemper virus (CDV) RNA polymerase were analyzed. The sequence was obtained from polymerase chain reaction (PCR) products using either c-DNA clones from a genomic library as template or in most instances genomic CDV RNA. Clones coding for the precise 3'- and 5'-ends of the CDV genome were sequenced and the results confirmed by additional PCR experiments. The virtual identity of terminal sequences and spacing at the two noncoding ends speak to the importance of these areas in replication and transcription. The sequence for each of the CDV gene boundaries was defined and all were compared to related viruses. This report completes the sequence determination of the CDV genome.

Base Sequence↗
PubMed1990

Nucleotide sequence and nuclear protein binding of the two regulatory sequences upstream of the am (GDH) gene in Neurospora.

We have constructed a series of deletions in the 5' non-coding sequences of the cloned Neurospora crassa am gene which specifies NADP specific glutamate dehydrogenase. All of the deletions begin at -4.4 kb with respect to the am transcription start site and extend for various distances toward the am gene. Using vectors with a truncated fragment of the am gene, we introduced these deletions into the chromosome upstream of am by transformation. Analysis of glutamate dehydrogenase expression in strains with the deletion mutations confirmed that there are two upstream regulatory sequences (URS) that control the expression of the am gene. The more distal of these elements (URSam beta) has been limited to the 157 bp between -1924 and -2081 with respect to the start of am transcription. The proximal element (URSam alpha) was limited to the 97 bp between -1296 and -1393. The DNA sequence of the entire region was determined. Within the sequences that contain the URS elements several regions of homology with yeast UAS sequences were found. Gel mobility assays with DNA fragments containing the URS elements indicated that sequences in both elements are bound by nuclear proteins from Neurospora. The interaction of these proteins and the DNA fragments was found to be specific.

Base Sequence↗
PubMed1987

A plasmid sequence from Rhizobium leguminosarum 300 contains homology to sequences near the octopine TL-DNA right border.

The DNA sequence from a Rhizobium leguminosarum 300 (RL300) plasmid that contains homology to the Tc-DNA of Agrobacterium tumefaciens is described. The RL300 sequence has 78% homology to a 359 bp sequence in the Tc-DNA of pTi15955. The RL300 homology starts approximately 100 bp from the 24 bp border sequence of the TL-DNA and ends approximately 3 bp from an IS66 homolog in the Tc-DNA. An unusual feature of the RL300 homology is the presence of 81 bp direct repeats with Tc-DNA homology, separated by 201 bp. One end of each direct repeat has a 12 bp palindrome. Four cloned sequences of RL300 with homology to the T DNA region were hybridized to plasmid lysates of RL300 derivatives to determine the source of each plasmid. The sequenced homolog, originally on pRH228, was isolated from pRL7JI; the other 3 homologs were isolated from the transmissable plasmids pRL7JI (pRH235) and pRL8JI (pRH235 and pRH236).

Amino Acid Sequence↗
PubMed1993

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences.

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence for Campylobacter jejuni subsp. jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.

Base Sequence↗
PubMed1990

Direct sequencing of a HLA-DRB gene by polymerase chain reaction: sequence variation in DRw8 specificity.

The nucleotide sequence of a HLA-DRB gene with a predominant subtype of DRw8 specificity in Japanese (DR8.1) was determined with single-stranded DNA enzymatically amplified by polymerase chain reaction (PCR). The sequence differs at a single amino acid from both of the published DRw8/Dw8.1 and DRw8/Dw8.2 sequences: isoleucine67(AUC) instead of phenylalanine67(TTC) in DRw8/Dw8.1 and serine57(AGC) instead of aspartic acid57(GAT) in DRw8/Dw8.2. On the other hand the DR8.1 and DRw8/Dw8.3 have the same amino acid sequence although one silent nucleotide substitution has occurred between the two sequences. These results indicate that Japanese DR8.1 specificity corresponds to DRw8/Dw8.3. Furthermore, an oligonucleotide probe specific for this sequence was synthesized and hybridized with 33 HLA-typed controls. This probe clearly distinguished the particular subtype from other DRw8 subtypes and specificities.

Amino Acid Sequence↗
PubMed1989

DNA sequence of a class I pseudogene from the Tla region of the murine MHC: recombination at a B2 alu repetitive sequence.

We have determined the DNA sequence of a BALB/c Tla region class I gene from the major histocompatibility complex (MHC) that had been identified previously as encoding a murine antigen by DNA-mediated gene transfer. Analysis of the DNA sequence shows, however, that this gene, the T1c gene from the Tlac genotype, could not encode a TL antigen or any other functional class I molecule due to the presence of numerous stop codons and frameshift mutations in the coding regions. This result suggests that the earlier transformation data may have been incorrect or perhaps that the clone containing the T1c gene contains sequences that induced expression of a serologically reactive Tla gene in the genome of the recipient L cell. The T1c gene is structurally related to the previously sequenced T13c gene that encodes a serologically defined TL antigen. The 3' half of the T1c gene including exons 4, 5, 6, and the 3' untranslated region has about 85% nucleotide similarity (including introns) with the corresponding parts of the T13c gene; however, the 5' half of the T1c gene has little homology with the T13c gene. There is a sharp line of demarcation between the homologous and nonhomologous regions, and this border occurs precisely at a B2 Alu repeat sequence present in the T13c gene. This suggests that a recombination event took place here and that an Alu repeat sequence that is known to have characteristics of transposable elements played some role in a recombination or gene conversion event.

Animals↗
PubMed2003

A molecular study of Eubothrium rugosum (Batsch, 1786) (Cestoda: Pseudophyllidea) using ITS rDNA sequences, with notes on the distribution and intraspecific sequence variation of Eubothrium crassum (Bloch, 1779).

The internal transcribed spacers (ITS-1 and ITS-2) of the ribosomal DNA (rDNA) of fish cestodes of the genus Eubothrium were sequenced. The ITS sequences of Eubothrium rugosum were determined and compared with previously analysed congeneric species, Eubothrium crassum and Eubothrium salvelini. The ITS-1 sequences of E. rugosum and E. crassum were 535 bp long, the length of E. salvelini ITS-1 was 536 bp. The ITS-2 region was found to be 403 bp in E. rugosum and E. crassum, and 401-402 bp in E. salvelini. The ITS-1 region of E. rugosum was closer to E. salvelini (identity 98.9-99.1%) than to E. crassum(97.9-98.1%), while the sequence similarity within the ITS-2 region was almost identical (97.5-98.0% for E. crassum; 97.7-98.3% for E. salvelini). Several restriction enzymes were found to be suitable for the differentiation of the three Eubothrium species by PCR-RFLP. The intraspecific sequence variation of E. crassum from different fish hosts (freshwater and marine) and European regions was very low, 0.2% for ITS-1 and 0.5% for ITS-2. Analysis of the ITS sequences of specimens from rainbow trout from three localities in Scotland revealed that both E. crassum and E. salvelini are present in this fish host.

Animals↗
PubMed1991

Multiplex polymerase chain reaction amplification and direct sequencing of homologous sequences: point mutation analysis of the ras genes.

ras proto-oncogenes are activated by point mutation in a wide variety of human and animal tumors, making ras gene analysis a major area of clinical and basic cancer research. Activating point mutations, in each of the three ras genes (Ha-, Ki-, or N-ras), usually occur in one of three specific codons (12, 13, or 61). Thus, an adequate assessment of activating ras gene mutations should include the analysis of at least nine codons. We have developed a rapid method for point mutation analysis of the ras genes, which involves simultaneous (multiplex) PCR amplification of all three homologous ras genes (in the regions surrounding codons 12-13 and codon 61) in a single reaction starting with only 1 microgram of genomic DNA. Although multiplex PCR has been previously used for unrelated sequences, we demonstrate here that multiplex PCR can also be used for highly homologous sequences. Importantly, after coamplification, each of the homologous ras genes can be individually and specifically sequenced even though the other two closely related genes are present in the same template mixture, by using high-stringency conditions permitted by Taq DNA polymerase. An automated multicycle DNA sequencing procedure is used to allow the double-stranded PCR products to be sequenced directly without the need to generate single-stranded templates, further simplifying the protocol. Our multiplex PCR amplification and direct DNA sequencing procedures should greatly facilitate more complete analyses of activating ras gene point mutations, particularly in studies involving many tumor samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Amino acids 356-372 constitute a Gi-activator sequence of the alpha 2-adrenergic receptor and have a Phe substitute in the G protein-activator sequence motif.

The human alpha 2-adrenergic receptor contains the sequence KASRWRGRQNREKRFTF (amino acids 356-372) at the C-terminal end of its third intracellular loop. This sequence satisfies the structural criteria for G protein-activating sequences [(1992) J. Biol. Chem. 267, 8342-8346] except that the C-terminal sequence is B-B-X-X-Phe instead of B-B-X-B or B-B-X-X-B (B: basic residue, X: non-basic residue). Nevertheless, the synthetic peptide corresponding to this sequence (peptide alpha 2-F) was found to activate Gi and Go strongly with a saturated effect at 1-3 microM. Furthermore, the substitution of the C-terminal Phe of peptide alpha 2-F with Arg, Trp, and Tyr (but not Ala or Asp) did not appreciably affect the Gi-activating potency. It is suggested that the C-terminal basic residue of the B-B-X-X-B motif in Gi-activating sequences can be replaced by an aromatic residue.

Amino Acid Sequence↗
PubMed1985

Genomic organization and nucleotide sequence of two distinct histone gene clusters from Xenopus laevis. Identification of novel conserved upstream sequence elements.

We have performed a detailed analysis of the genomic organization and the nucleotide sequence of two distinct Xenopus laevis histone gene clusters totaling approximately 23.5 X 10(3) base-pairs. Each cluster contains at least one copy of each of the five histone genes. However, these genes are present in different arrangements within each cluster and different H1A, H2A and H2B proteins (variants) are encoded by the respective genes of each cluster. Southern blot analysis of genomic X. laevis DNA indicates that each cluster is a member of a distinct family of tandemly repeated histone gene clusters. A comparative analysis of the nucleotide sequences flanking the histone genes within these two clusters has revealed the presence of multiple conserved sequence elements that are specific for each histone gene class and located at preferred upstream positions. Several of these elements correspond to sequences that are known to be required for maximal transcription of the corresponding genes. Most of these sequence elements have not been identified previously, although we find that many of them are present at corresponding locations upstream of histone genes from other organisms. We suggest that the conserved upstream sequence elements may play an important role in the expression of histone genes in vivo.

Amino Acid Sequence↗
PubMed1983

Ross River virus 26 s RNA: complete nucleotide sequence and deduced sequence of the encoded structural proteins.

The complete sequence of the 26 S RNA of Ross River virus (T48 strain) has been obtained and from this the amino acid sequences of the encoded structural proteins have been deduced. These include a basic capsid protein and two envelope glycoproteins. The nucleotide sequence was obtained by chemical sequence analysis of both single-stranded and double-stranded cDNA made to RNA and the sequence data so obtained was rapidly aligned by making use of the protein homology found among the alphaviruses. The polyprotein precursor encoded by the 26 S RNA of Ross River virus is 75% homologous to that of Semliki Forest virus and 48% homologous to that of Sindbis virus. The extent of homology is not uniform within a protein or between proteins and this is discussed with respect to the possible function of the various polypeptide domains in the virus life cycle. In each case the putative attachment site of the amino proximal carbohydrate chains of the three glycoproteins is conserved, whereas the attachment site of a second chain, if present, is not conserved. The 3'-untranslated region of Ross River virus RNA is 524 nucleotides long. It contains a sequence of about 50 nucleotides in length which is present in four copies but which is not shared with other alphaviruses examined.

Alphavirus↗
PubMed1985

Nucleotide sequence of the rpsU-dnaG-rpoD operon from Salmonella typhimurium and a comparison of this sequence with the homologous operon of Escherichia coli.

In Escherichia coli the genes encoding ribosomal protein S21 (rpsU), DNA primase (dnaG), and the 70-kDal sigma subunit of RNA polymerase (rpoD) are contained in a single operon. These gene products are involved in the initiation of translation, DNA replication, and transcription, respectively. We have examined the homologous region in the closely related bacterium Salmonella typhimurium and have found that the same three genes are similarly organized. We have sequenced the DNA for this operon in S. typhimurium and have compared the (nt) nucleotide and amino acid (aa) sequences with E. coli. In the coding regions, the sequence conservation varies from extremely high for rpsU to moderate for dnaG with respect to both nt and aa sequence. In the noncoding regions, sequences thought to be important for the regulation of transcription are conserved, while other sequences are not conserved. aa differences in DNA primase and sigma are not randomly distributed and suggest regions that may be important for protein structure or function.

Amino Acid Sequence↗
PubMed1999

Occurrence of heavy chain of 7S IgM half-molecule whose NH2-terminal sequence is identical with that of kappa light chain sequence in patients with Waldenstrom macroglobulinemia.

We report a rare case of a half molecule 7S IgM (HM 7SIgM) consisting of a unique mu heavy chain and kappa light chain found in blood and urine samples from a patient with primary Waldenstrom macroglobulinemia. A 64kDa abnormal immunoglobulin was detected in serum and urine by immunoblot method, and purified by a two-dimensional SDS-PAGE after separation from IgG and albumin fractions on gel filtration. NH2-terminal amino acid sequence analysis of the heavy chain revealed that residues 1-20 were identical to those of the NH2-terminal region of kappa light chain derived from the same patient. This sequence was then followed by a sequence that could not be identified by a computer homology search on the protein database. Using polypeptide segments obtained from the unique mu chain by digestion with endopeptidase, we identified a sequence spanning from residue 127 in the variable region of the known mu chain to residue 19 in the known CH1 domain and a sequence spanning from residues 67-82 in the heavy chain variable region class II. From these results, we concluded that the 64 kDa protein was an abnormal half molecule 7S IgM consisting of a kappa light chain and a unique mu heavy chain of 35 kDa polypeptide in which the NH2-terminal 20 amino acids were replaced by 20 amino acids derived from the sequence of kappa light chain in the NH2-terminal region.

Amino Acid Sequence↗
PubMed1987

A quantitative approach to sequence comparisons of nitrogenase MoFe protein alpha- and beta-subunits including the newly sequenced nifK gene from Klebsiella pneumoniae.

The nucleotide sequence was determined for part of the Klebsiella pneumoniae nif gene cluster containing the 3' end of the nifD gene and the entire length of the nifK gene (encoding the alpha- and beta-subunits of the nitrogenase MoFe protein respectively), as well as the putative start of the nifY gene, a gene of as yet unknown function. A broad-based comparison of a number of MoFe protein alpha-subunits, beta-subunits and alpha-versus beta-subunits was carried out by the use of a computer program that simultaneously aligns three protein sequences according to the mutation data matrix of Dayhoff. A new kind of quantitative statistical measure of the similarity between the aligned sequences was obtained by calculating and plotting standardized similarity scores for overlapping segments along the aligned proteins. This calculation determines if a test sequence is similar to the consensus sequence of two other proteins that are known to be related to each other. The different beta-subunits compared were found to be significantly similar along most of their sequence, with the exception of two relatively short regions centred around residues 225 and 300, which contain insertions/deletions. The overall pattern of similarity between different alpha-subunits exhibits resemblance to the overall pattern of similarity between different beta-subunits, including regions of low similarity centred around residues 225 and 340. Comparison of alpha-subunits with beta-subunits showed that a region of significant similarity between the two types of subunits was located approximately between residues 120 and 180 in both subunits, but other parts of the proteins were only marginally similar. These results provide insights into likely tertiary structural features of the MoFe protein subunits.

Amino Acid Sequence↗