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At least 793 records · Page 44Linked to original sources
PubMed1989

Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae.

To clarify carbon source-dependent control of the glycolytic pathway in the yeast Saccharomyces cerevisiae, we have initiated a study of transcriptional regulation of the pyruvate kinase gene (PYK). By deletion analysis of the 5'-noncoding region of the PYK gene, we have identified an upstream activating sequence (UASPYK1) located between 634 and 653 nucleotides upstream of the initiating ATG codon. The promoter activity of the PYK 5'-noncoding region was abolished when the sequence containing the UASPYK1 was deleted from the region. Synthetic UASPYK1 (26mer), in either orientation, was able to restore the transcriptional activity of UAS-depleted mutants when placed upstream of the TATA sequence located at -199 (ATG as +1). While the UASPYK1 was required for basal to intermediate levels of transcriptional activation, a sequence between -714 and -811 was found to be necessary for full activation. On the other hand, a sequence between -344 and -468 was found to be responsible for transcriptional repression of the PYK gene when yeast cells were grown on nonfermentable carbon sources. This upstream repressible sequence also repressed transcription, although to a lesser extent, when glucose was present in the medium. The possible mechanism for carbon source-dependent regulation of PYK expression through these cis-acting regulatory elements is discussed.

Base Sequence↗
PubMed1989

Nucleotide sequence of the DNA complementary to avian (chicken) preproparathyroid hormone mRNA and the deduced sequence of the hormone precursor.

The nucleotide sequence of avian (chicken) prepro-PTH (prepro-PTH) mRNA was determined from a 2.3-kilobase fragment of complementary chicken parathyroid DNA cloned in E. coli MM 924. Northern blot analysis of chicken parathyroid mRNA, using both bovine and chicken cDNA probes, showed that the mRNA (2.3 kilobases) for chicken hormone precursor was approximately 3 times the size of mRNA for mammalian prepro-PTH. Cleavage of the cloned DNA with restriction endonuclease Pstl resulted in three fragments, each of which was subjected to sequence determination. The hormone sequence deduced from the DNA showed that chicken prepro-PTH mRNA encoded a 119-amino acid precursor which included a 25-amino acid signal sequence, a six-residue prohormone peptide, and an 88-amino acid hormone. The hormonal peptide was four residues longer than all known mammalian homologs and included gene deletions and insertions. There was significant homology of sequence in the biologically active 1-34 region with mammalian hormones, but much less in the middle and carboxyl-terminal regions. This is the first nonmammalian PTH sequence to be determined and should prove useful in studying evolution of the gene as well as structure-function relationships of the hormone.

Animals↗
PubMed2006

The regulatory function of the upstream sequence of the beta-conglycinin alpha subunit gene in seed-specific transcription is associated with the presence of the RY sequence.

beta-conglycinin, a major component of seed-storage proteins in soybean, comprises three subunits: alpha, alpha', and beta. Expression of these genes is spatially regulated in a stringent manner and occurs during seed development. To understand the mechanisms that control expression of the alpha subunit gene, we analyzed the nucleotide sequence of the 2.9-kb region upstream of the gene. The upstream sequence up to -1357 or a series of its 5'-deleted derivatives was fused to the beta-glucuronidase (GUS) gene. These reporter gene constructs were introduced into Arabidopsis thaliana plants via Agrobacterium-mediated gene transfer. Prominent GUS activity was detected in developing seeds of the T3 generation when 245 bp or longer sequences of the upstream region were fused to the GUS gene. We found a clear association of decreased GUS activity with a stepwise deletion of a region containing the RY sequence from the original construct. These results are consistent with the notion that multiple sequence elements including the RY sequences are involved in the seed-specific transcriptional activation of the beta-conglycinin alpha subunit gene in soybean.

Antigens, Plant↗
PubMed2001

Cloning of deleted sequences (CODE): A genomic subtraction method for enriching and cloning deleted sequences.

The deletion of specific genomic sequences is believed to influence the pathogenesis of certain diseases such as cancer. Identification of these sequences could provide novel therapeutic avenues for the treatment of disease. Here, we describe a simple and robust method called cloning of deleted sequences (CODE), which allows the selective cloning of deleted sequences from complex human genomes. Briefly, genomic DNA from two sources (human normal and tumor samples) was digested with restriction enzymes (e.g., BamHI, BglII, and BclI), then ligated to special linkers, and amplified by PCR. Tester (normal) DNA was amplified using a biotinylated primer and dNTPs. Driver (tumor) DNA was amplified using a non-biotinylated primer, but with dUTP instead of d7TP After denaturation and hybridization, all the driver DNA was destroyed with uracil-DNA glycosylase (UDG), and all imperfect hybrids were digested with mung bean nuclease. Sequences deleted from the driver DNA but present in the tester DNA were purified with streptavidin magnetic beads, and the cycle was repeated three more times. This procedure resulted in the rapid isolation and efficient cloning of genomic sequences homozygously deleted from the driver DNA sample, but present in the tester DNA fraction.

Base Sequence↗
PubMed1993

A general method for routine sequencing of cloned DNA fragments using commercial dye primers and its application in sequence analysis of Toxoplasma gondii RH genome fragments.

A generally applicable approach for amplification and subsequent sequencing using commercially available dye primers is described. The polymerase chain reaction primers, one of which is biotinylated, are tagged at their 5' end with sequences of commercial sequencing primers. After purification and strand separation on streptavidin-coated magnetic beads, both strands are sequenced automatically on an Applied Biosystems 373A DNA sequencer using the appropriate standard dye-labeled sequencing primers. This approach is demonstrated on PstI and MnlI DNA fragments from Toxoplasma gondii RH cloned in the in-house developed derivatives of vector pJRtac99.

Animals↗
PubMed2000

Direct and reversed amino acid sequence pattern analysis: structural reasons for activity of reversed sequence sites and results of kinase site mutagenesis.

During studies of kinase phosphorylation, not all functional kinase phosphorylation may be found using consensus sequence patterns. This type of phosphorylation is termed 'non-consensus' or 'cryptic' phosphorylation. Results presented here based on molecular dynamics of short peptides show that protein kinases may phosphorylate not only established consensus sequences (reading a sequence from N-terminus to C-terminus) but also reversed consensus sequences (reading from C- to N-terminus). Several protein sequences were analysed and corresponding biochemical data were presented. Similarity of molecular shapes of direct and reversed consensus peptides, and sequence conservation in the regions of reversed sites in the analysed proteins, indicate that at least part of the phosphorylation sites considered as 'cryptic' may be explained in terms of reversed consensus pattern occurrences.

ATP-Binding Cassette Transporters↗
PubMed1989

Sequence analysis of a human gene responsible for drug resistance: a rapid method for manual and automated direct sequencing of products generated by the polymerase chain reaction.

We developed a system for rapid, manual and automated sequence analysis by utilizing and modifying methods used in conjunction with the polymerase chain reaction (PCR). We are using these techniques to detect single base mutations in the dihydrofolate reductase (DHFR) gene giving rise to methotrexate (MTX) resistance of tumor cells obtained from patients with malignancies. Amplifying in vitro both genomic DNA and transcripts of the human DHFR we are able to reproducibly generate single-stranded templates. Utilizing [alpha-35S]dATP and both the universal and reverse sequencing primers we obtain sequence information from either strand. The methods described have been successfully used for automated sequencing with the Applied Biosystems Model 370A Sequencer using both modified T7 DNA polymerase and Taq I. DNA polymerase for dideoxy-termination sequencing. The use of this methodology to detect a single base change in a human colon carcinoma cell line, HCT-8, is illustrated.

Adenocarcinoma↗
PubMed1985

Sequence of human asialoglycoprotein receptor cDNA. An internal signal sequence for membrane insertion.

A cDNA library from the human hepatoma cell line Hep G2 was prepared in the expression vector lambda gt11. Using specific antibodies, a cDNA clone containing the entire coding sequence for the human asialoglycoprotein receptor was isolated and sequenced. The deduced amino acid sequence of 291 residues is very homologous to the sequence of the major asialoglycoprotein receptor protein from rat. The comparison shows that there is no significant post-translational processing and no leader sequence, cleaved or uncleaved, at the amino terminus. An internal signal sequence, probably the membrane-spanning segment, residues 41-59, is assumed to direct insertion of the carboxyl-terminal ligand binding portion of the receptor across the endoplasmic reticulum membrane.

Amino Acid Sequence↗
PubMed1987

Rat liver NAD(P)H: quinone reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the amino acid sequence of the corresponding protein.

We have determined the nucleotide sequence of a cDNA clone, pDTD55, complementary to rat liver quinone reductase mRNA (Williams, J.B., Lu, A.Y.H., Cameron, R.G., and Pickett, C.B. (1986) J. Biol. Chem. 261, 5524-5528). The cDNA clone contains an open reading frame of 759 nucleotides encoding a polypeptide comprised of 253 amino acids with a Mr = 28,564. To verify the predicted amino acid sequence of quinone reductase, we have been able to align the amino acid sequences of a cyanogen bromide digest of the purified enzyme to the sequence deduced from the cDNA clone. A comparison of the quinone reductase sequence with other known flavoenzymes did not reveal a significant degree of amino acid sequence homology. These data suggest that the quinone reductase gene has evolved independently from genes encoding other flavoenzymes.

Amino Acid Sequence↗
PubMed1987

Cloning and sequence analysis of cDNAs encoding mammalian cytosolic malate dehydrogenase. Comparison of the amino acid sequences of mammalian and bacterial malate dehydrogenase.

A cDNA clone, named ppcMDH-1 and covering a part of the coding region for the porcine cytosolic malate dehydrogenase (cMDH) mRNA, was isolated from a porcine liver cDNA library. Subsequently, mouse cMDH cDNA clones were isolated from mouse liver and heart cDNA libraries, using the ppcMDH-1 cDNA as a probe. The longest clone, named pmcMDH-5, was sequenced and the primary structure of the mouse cMDH deduced from its cDNA sequence showed that the mouse cMDH consists of the 334-amino acid residues. When the amino acid sequence of the mouse cMDH was compared with that of the porcine cMDH, they shared a 93% homology. On the other hand, the amino acid sequences of mouse cMDH and mitochondrial MDH (mMDH) showed about 23% overall homology. Surprisingly, comparison of the amino acid sequences among the mammalian and bacterial MDHs revealed that the homology between the mouse cMDH and thermophilic bacterial MDH, as well as the homology between the mouse mMDH and Escherichia coli MDH, markedly exceeds the intraspecies sequence homology between mMDH and cMDH from mice.

Amino Acid Sequence↗
PubMed1987

The amino acid sequence of Emu osteocalcin: gas phase sequencing of Gla-containing proteins.

Osteocalcin (OC), the major gamma carboxyglutamic acid (Gla)-containing protein of vertebrate bone, has been isolated from bones of the emu (Dromaius novaehollandae) and the primary structure determined by a combination of gas phase N-terminal sequencing of the intact molecule and a proteolytic fragment, and carboxypeptidase Y C-terminal sequencing. Gla residues were located by counting tritium radioactivity in fractions from the N-terminal sequencing of the tritiated/thermally decarboxylated molecule. Emu OC consists of 48 amino acid residues containing 3 Gla residues, and a single disulphide bond. The C-terminal 29 residues are identical to those of the human and sheep OC sequences. Alignment of the N-terminal sequence against those of other OCs reveals greater sequence homology with chicken OC than with mammalian OCs.

1-Carboxyglutamic Acid↗
PubMed1987

cDNA cloning and sequencing reveals that the electrophoretically constant DR beta 2 molecules, as well as the variable DR beta 1 molecules, from HLA-DR2 subtypes have different amino acid sequences including a hypervariable region for a functionally important epitope.

Two-dimensional gel electrophoresis (2D-PAGE) of DR molecules from three different Dw subtypes (Dw2, Dw12, and FJO) of the HLA-DR2 haplotype reveals that at least two DR beta genes are expressed. Protein mixing experiments demonstrate that one of the two expressed DR beta molecules is electrophoretically variable (referred to as DR beta 1), and the other (DR beta 2) migrates constantly among DR2 subtypes. We have constructed cDNA libraries from Dw12 and FJO homozygous typing cells (HTC DHO for Dw12 and HTC FJO for FJO) and isolated DR beta cDNA clones. Four of these clones (FJO-13, DHO-8, FJO-6, and DHO-7) were sequenced, and the deduced amino acid sequences were compared with each other and with two published amino acid sequences for the DR beta molecules derived from a DR2-Dw2HTC. Prediction of the migration patterns on 2D-PAGE from the amino acid sequences of these and other DR beta molecules allows the tentative designation of the two full-length cDNA (DHO-8 and FJO-13) as coding for DR beta 2 molecules and the other two cDNA (DHO-7 and FJO-6) for DR beta 1 molecules. Amino acid sequence comparisons also show that the constantly migrating DR beta 2 molecules, as well as the electrophoretically variable DR beta 1 molecules, from Dw2, Dw12, and FJO have different primary amino acid sequences, including a clustered difference in the third hypervariable region of the polymorphic first domain.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the alpha subunit of bacterial luciferase.

The nucleotide sequence of the 1.85-kilobase EcoRI fragment from Vibrio harveyi that was cloned using a mixed-sequence synthetic oligonucleotide probe (Cohn, D. H., Ogden, R. C., Abelson, J. N., Baldwin, T. O., Nealson, K. H., Simon, M. I., and Mileham, A. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 120-123) has been determined. The alpha subunit-coding region (luxA) was found to begin at base number 707 and end at base number 1771. The alpha subunit has a calculated molecular weight of 40,108 and comprises a total of 355 amino acid residues. There are 34 base pairs separating the start of the alpha subunit structural gene and a 669-base open reading frame extending from the proximal EcoRI site. At the 3' end of the luxA coding region there are 26 bases between the end of the structural gene and the start of the luxB structural gene. Approximately two-thirds of the alpha subunit was sequenced by protein chemical techniques. The amino acid sequence implied by the DNA sequence, with few exceptions, confirmed the chemically determined sequence. Regions of the alpha subunit thought to comprise the active center were found to reside in two discrete and relatively basic regions, one from around residues 100-115 and the second from around residues 280-295.

Amino Acid Sequence↗
PubMed1983

Amino acid sequence of alpha-amylase from Bacillus amyloliquefaciens deduced from the nucleotide sequence of the cloned gene.

We have isolated by molecular cloning the gene coding for the alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1.) from Bacillus amyloliquefaciens and determined its complete nucleotide sequence. The gene cloned in the plasmid pUB110 using Bacillus subtilis as a host, was contained in a 2.3-kilobase insert. Starting from an ATG initiator codon, an open reading frame comprising a total of 514 amino acids (1542 base pairs) was found within the cloned DNA fragment. The gene region encoding the COOH terminus of alpha-amylase was located by direct COOH-terminal analysis of the purified exoenzyme. The NH2-terminal portion of the gene encodes a 31 amino acid-long signal peptide (Palva, L., Pettersson, R. F., Kalkkinen, N., Lehtovaara, P., Sarvas, M., Söderlund, H., Takkinen, K., and Kääriäinen, L. (1981) Gene 15, 43-51). Since the signal peptide is correctly cleaved in the new host, as shown here by direct NH2-terminal sequence analysis, the exoamylase consists of 483 amino acid residues, corresponding to a molecular weight of 54,778. The reading frame used to deduce the amino acid sequence was found to be correct by comparison with partial amino acid sequence data published previously (Detera, S. D., and Friedberg, F. (1979) Int. J. Peptide Protein Res. 14, 364-372; Chung, H., and Friedberg, F. (1980) Biochem. J. 185, 387-395). Several differences between the sequence presented here and the partial ones published previously, however, were found. The nucleotide sequences both 5' and 3' to the alpha-amylase gene revealed palindromic structures including a stretch of six T-residues, suggesting transcription termination signals on both sides of the gene. Thus, it appears that alpha-amylase is translated from a monocistronic mRNA.

Amino Acid Sequence↗
PubMed1981

Amino acid sequence of the gene 0.3 protein of bacteriophage T7 and nucleotide sequence of its mRNA.

The amino acid sequence of purified gene 0.3 protein of T7, the protein responsible for overcoming host restriction, has been determined. The nucleotide sequence of the 0.3 RNA, the messenger RNA that codes for both the 0.3 protein and the gene 0.4 protein, a T7 protein of unknown function, has also been determined. The 0.3 RNA is 578 nucleotides long, 509 of which are used to code for the 2 proteins. The coding sequences do not overlap, but the termination codon for the 0.3 protein and the presumed initiation codon for the 0.4 protein do overlap in the sequence UAAUG. The 0.3 protein is very acidic: 34 of its 116 amino acids are aspartic or glutamic acid and only 6 are arginine or lysine. The 0.3 protein contains no cysteine. The nucleotide sequence predicts that the 0.4 protein consists of 50 amino acids and contains no histidine or proline. The effects of different mutations indicate that a protein which contains only the first 87 amino acids of the 0.3 protein is unable to prevent host restriction in vivo; one that contains te first 93 amino acids has weak function; and one that has the first 94 amino acids (plus 2 that are not in the wild type sequence) is fully able to prevent host restriction. The apparently critical 94th amino acid is tryptophan. The mutant 0.3 proteins that contain 87 or more amino acids appear to be reasonably stable in vivo, but those that contain 78 or fewer are apparently too unstable to have been observed by gel electrophoresis.

Amino Acid Sequence↗
PubMed1982

Rat pancreatic ribonuclease messenger RNA. The nucleotide sequence of the entire mRNA and the derived amino acid sequence of the pre-enzyme.

We have cloned via recombinant DNA technology the mRNA sequence of rat pancreatic ribonuclease, and have determined the entire nucleotide sequence of the mature message. Clones bearing RNase sequences within a double-stranded complementary DNA library of rat pancreatic mRNA were initially detected by hybridization with size-fractionated rat pancreatic polyadenylated RNA that included mRNA 0.85 to 1.0 kilobase in length. Recombinant plasmids bearing RNase mRNA sequences were conclusively identified by comparison of the amino acid sequence of the encoded protein with the known amino acid sequence of rat RNase. RNase mRNA is 783 nucleotides in length, plus a poly(a) tail with an average length of 140 nucleotides, and contains long 5' and 3' noncoding regions relative to other pancreatic mRNAs. It encodes a secretory preRNase of 152 amino acid residues including a signal peptide of 25 amino acids.

Amino Acid Sequence↗
PubMed1994

The sequence of 12.5 kb from the right arm of chromosome II predicts a new N-terminal sequence for the IRA1 protein and reveals two new genes, one of which is a DEAD-box helicase.

We have determined the complete nucleotide sequence of a 12.5 kb segment from the right arm of chromosome II carried by the cosmid alpha 20. The sequence encodes the 5' end of the IRA1 gene. Two complete new open reading frames and the 3' non-coding region of the SUP1 (SUP45) gene. A comparison of our sequence with the data bank reveals a 154 amino acid extension at the N-terminus of Ira1p compared to the previously predicted sequence. According to the 11th edition of the Saccharomyces cerevisiae genetic map, our sequence should encode the MAK5 gene, which is necessary for the maintenance of dsRNA killer plasmids. One of the two new open reading frames, YBR1119, is predicted to encode an RNA helicase, thus YBR1119 may correspond to the MAK5 gene.

Amino Acid Sequence↗
PubMed1995

Sequence analysis of frog alpha B-crystallin cDNA: sequence homology and evolutionary comparison of alpha A, alpha B and heat shock proteins.

alpha-Crystallin is a major lens protein present in the lenses of all vertebrate species. Recent studies have revealed that bovine alpha-crystallins possess genuine chaperone activity similar to small heat-shock proteins. In order to facilitate the determination of the primary sequence of amphibian alpha B-crystallin, cDNA encoding alpha B subunit chain was amplified using a new "Rapid Amplification of cDNA Ends" (RACE) protocol of Polymerase Chain Reaction (PCR). PCR-amplified product corresponding to alpha B subunit was then subcloned into pUC18 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the automatic fluorescence-based dideoxynucleotide chain-termination method. Sequencing more than five clones containing DNA inserts coding for alpha B-crystallin subunit constructed only one complete full-length reading frame of 522 base pairs similar to that of alpha A subunit, covering a deduced protein sequence of 173 amino acids including the universal translation-initiating methionine. The frog alpha B crystallin shows 69, 66 and 56% whereas alpha A crystallin shows 83, 81 and 69% sequence similarity to the homologous chains of bovine, chicken and dogfish, respectively, revealing a more divergent structural relationship among these alpha B subunits as compared to alpha A subunits. Structural analysis and comparison of alpha A- and alpha B-crystallin subunits from eye lenses of different classes of vertebrates also shed some light on the evolutionary relatedness between alpha B/alpha A crystallins and the small heat-shock proteins.

Amino Acid Sequence↗