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At least 523 records · Page 29Linked to original sources
PubMed1994

Mosaic structure of large regions of the Lactococcus lactis subsp. cremoris chromosome.

Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris are closely related phenotypically and genetically. Here we report that certain regions of their chromosomes diverge considerably more than others. Conserved regions differ by less than 20%, whilst variable regions differ by more than 60%. This mosaic structure may have arisen by horizontal gene transfer from distantly related bacteria since in a particular region of the L. lactis subsp. cremoris chromosome the G+C content and the codon bias are not typical for lactococci. Such an exchange, which conserves the function of the gene and cannot be achieved under selective pressure, may be of considerable importance in the evolution of bacteria.

Amino Acid Sequence↗
PubMed1969

Structural determinant of a ribosomal protein: K locus.

One of the ribosomal proteins (30S-8B) of Escherichia coli B has a greater electrophoretic mobility than the homologous protein (30S-8K) in E. coli K. The amino acid compositions, tryptic peptides, and cyanogen bromide peptides of these two homologous proteins unambiguously indicate that the genetic locus which determines the electrophoretic mobility of protein 30S-8 is the structural gene for this protein.

Amino Acid Sequence↗
PubMed1991

Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein.

We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag.

Base Sequence↗
PubMedDate not supplied

Comparison of functional peptide encoded in the Escherichia coli 23S rRNA with other peptides involved in cis-regulation of translation.

A new approach for studying functional rRNA fragments has been developed based on using a plasmid library expressing random fragments of rRNA. A 34 nucleotide long fragment of Escherichia coli 23S rRNA has been identified that renders cells resistant to erythromycin, when expressed in vivo. The rRNA fragment contains a five codon long open reading frame, initiating at GUG and terminating at UAA, with a Shine-Dalgarno sequence located at an appropriate distance from the initiator codon. Translation of this mini-gene is required for the observed erythromycin resistance. Experiments with in vitro translated, or synthetic, peptide indicate the ribosome as a likely target for the action of the identified rRNA-encoded peptide, which apparently remains associated with the ribosome after completion of its translation. The known properties of the rRNA-encoded peptide are compared with information about other functionally active short peptides that can be involved in regulation of translation.

Amino Acid Sequence↗
PubMed1984

Nucleotide sequence of rat liver aldolase B messenger RNA.

The nucleotide sequence of messenger RNA encoding rat liver aldolase B has been determined by sequence analysis using recombinant cDNAs cloned in bacterial plasmids. The sequence contains part of the 5'-untranslatable region (68 nucleotides), the entire coding region (1092 nucleotides), and the complete 3'-untranslatable region (387 nucleotides), excluding the poly(A) tail. A potential ribosomal-binding site is located about 30 nucleotides upstream from the initiation codon. The amino acid sequence of rat liver aldolase B is composed of 364 amino acids and has 70% homology with rabbit muscle aldolase A.

Amino Acid Sequence↗
PubMed1992

Origin, structure, and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression of argK in transgenic tobacco.

Pseudomonas syringae pv. phaseolicola produces the tripeptide N delta(N'-sulfo-diaminophosphinyl)-ornithylalanyl-homoarginin e (phaseolotoxin), which functions as a chlorosis-inducing toxin in the bean halo blight disease by inhibiting ornithine carbamoyltransferase (OCT). The bacterium possesses duplicate OCT genes, one of which, argK, encodes a toxin-resistant enzyme (ROCT) and imparts resistance to phaseolotoxin. We sequenced the argK gene from strain NPS3121, defined its promoter region, analyzed its regulation, and characterized its transcripts. The gene probably originated from another organism, since it is very distantly related to the argF gene encoding the housekeeping toxin-sensitive OCT and has low G+C content compared with the bacterial genome as a whole and with other protein-coding genes from P. syringae pv. phaseolicola. Optimized alignments of 13 OCT sequences allowed us to define key residues that may be responsible for toxin resistance and to identify a distinct prokaryotic amino acid signature, in ROCT, which argues for a prokaryotic origin of argK. An in-frame fusion of the argK coding region with the chloroplast transit peptide segment of the pea rbcS gene was introduced in Nicotiana tabacum by Agrobacterium-mediated transformation. The presence of an ROCT activity in transgenic plants was demonstrated by in vitro and in vivo assays. Some plants were toxin resistant, suggesting that pathogen-derived resistance to the toxin should be feasible in the pathogen's host.

Amino Acid Sequence↗
PubMed1995

An extranuclear expression system for analysis of cytoplasmic promoters of yeast linear killer plasmids.

Based on the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis two new linear hybrid plasmids were constructed which consist of pGKL1, into which in addition to the previously developed cytoplasmically expressible LEU2* selectable marker a glucose dehydrogenase-encoding bacterial gene (gdh A) has been integrated. One of the hybrid plasmids carries the bacterial gene preceded by an arbitrarily placed cytoplasmic promoter (upstream conserved sequence) in front of the coding region (pRKL121). The other plasmid was constructed in such a way that the ATG start codon of the gdh A gene was fused in frame to the ATG start codon of the killer plasmid's open reading frame 5 (pRKL122). The structures of both linear hybrid plasmids were confirmed by restriction analysis, Southern hybridization, and sequencing of the junction sites. Yeast strains carrying either of the plasmids expressed the glucose dehydrogenase gene; however, expression of the in phase fused gene was 40-fold higher compared to the arbitrarily placed cytoplasmic promoter. In general, an in phase fusion was not required for expression, but efficiency is dramatically enhanced when the 5' noncoding sequences in front of the heterologous genes are the same as those found on the native killer plasmids. The developed system can serve as a reporter for determining the efficiency of the different cytoplasmic promoters present on both linear plasmids. Hybrid plasmids were stably maintained without selective pressure in K. lactis and they were transferred and expressed also in Saccharomyces cerevisiae.

Bacillus megaterium↗
PubMed1990

[Cloning, structure and expression of the full-size lon gene in Escherichia coli coding for ATP-dependent La-proteinase].

Assembly of the full Escherichia coli K-12 lon gene from the EcoRI--SphI fragment of the bacterial DNA ("modified" gene) cloned and sequenced earlier and the PstI fragment of the same DNA containing 3'-terminal region of the lon gene has been performed. Both "modified" and full genes showed all phenotype properties of lon gene. The complete nucleotide sequence of the gene (2770 bp) coding for the 784 amino acid sequence of protease La was determined. Location of catalytically active serine, histidine and aspartic acid residues was suggested, and ATP-binding site found. The lon gene and protease La structures we found are compared with those described independently and differences observed are discussed.

ATP-Dependent Proteases↗
PubMed1973

Reinitiation of a lac repressor fragment at a codon other than AUG.

52 Spontaneous nonsense mutants in the lac i gene of Escherichia coli were isolated and characterized. All mutants located early in the gene show negative complementation in vivo with a wild-type i gene in a recA diploid strain. In vitro studies show that those mutants that display negative complementing activity in vivo also make lac repressor fragments retaining inducer binding and immunological crossreactivity with wild-type repressor. Amino-acid sequence analysis of these fragments shows that they arise by reinitiation at internal sities of the i message after chain termination at a prior amber or ochre codon. There are at least two different internal reinitiation sites in the first 200 nucleotides of the translated part of the i message. The first site corresponds to the first internal in phase AUG codon, which specifies the methionine residue at position 42 of the repressor protein. This site can be activated by an amber codon, 45 nucleotides before the AUG codon. The second site is only 60 nucleotides past the first site and can be activated by an amber mutation derived from residue 60 of the protein. The second initiation codon specifies the amino-acid leucine in the wild-type repressor, but the reinitiated fragment shows an amino-terminal methionine residue at this position. Therefore, the second initiation site seems to involve an in vivo ambiguity of the genetic code in that the same codon can be translated into two different amino acids depending on the recognition of this codon during initiation (when methionine is inserted) or elongation of protein synthesis (when leucine is inserted). The possibility that a codon other than AUG can act as an initiation codon in vivo is discussed.

Adenine Nucleotides↗
PubMed1980

Nucleotide sequence of influenza virus RNA segment 8 indicates that coding regions for NS1 and NS2 proteins overlap.

The smallest RNA segment of influenza A viruses (vRNA segment 8) has recently been shown to code for two unrelated nonstructural proteins (NS1 and NS2) translated from separate mRNAs. Molecular weight considerations indicated that there might not be enough space on vRNA segment 8 for the two coding regions unless they overlap. We have recently cloned in bacterial plasmids several genes of an avian influenza A virus, fowl plague virus (EPV), and now present the complete nucleotide sequence of FPV RNA segment 8 largely determined from the cloned DNA. The DNA sequence predicts two open protein synthesis reading frames that can be translated into polypeptides of sizes similar to those of NS1 and NS2. The coding regions for these polypeptides overlap by the equivalent of 43-60 amino acids, the exact amount depending on which of several possible methionines initiates the synthesis of NS2.

Amino Acid Sequence↗
PubMed2000

Molecular cloning and partial characterisation of the mouse Cyp51 cDNA.

Northern analysis of the mouse testis and liver RNA show two types of lanosterol 14alpha-demethylase (CYP51) transcripts, somatic and a testis-specific transcript. At least two CYP51 mRNAs are expressed in liver and testis, while the smallest transcript is expressed only in testis. The partial mouse Cyp51 cDNA (1052 bp) was obtained by RT-PCR cloning and was also found in expressed sequence tag (EST) clones. Bacterial artificial chromosome (BAC) clones containing mouse Cyp51 gene were also found. The coding part of the partial mouse CYP51 is 93.9% homologous to the rat and 87.9% to the human CYP51 cDNAs. There is less than 2% variation between the CYP51 EST clones to the RT-PCR cloned partial cDNA and BAC clones.

Animals↗
PubMedDate not supplied

A deletion affecting several gene candidates is present in the Evergrowing peach mutant.

Evergrowing (EVG) peach is one of only two described mutants affecting winter dormancy in woody perennial species. EVG peach does not set terminal buds, cease new leaf growth, nor enter into a dormant resting phase in response to winter conditions. The EVG mutation segregates in F2 progeny as a single recessive nuclear gene. A local molecular genetic linkage map around EVG was previously developed using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers, and a bacterial artificial chromosome (BAC) contig that contains the EVG mutation was assembled. A MADS box coding open reading frame (ORF) was found in a BAC of this contig and used as a probe. The probe detected a polymorphism between the wild-type and mutant genomes, and the polymorphism is indicative of a deletion in EVG peach. The EVG gene region contained six potential MADS-box transcription factor sequences, and the deletion in EVG affected at least four of these. The deletion was bracketed using RFLP analysis, which showed that it is contained within a segment of the genome no greater than 180 kb.

Base Sequence↗
PubMed1997

Biophysical investigations on the myb-DNA system.

The oncogene product c-myb is a transcriptional modulator and is known to play important roles in cell growth and differentiation. It binds to DNA in a sequence specific manner and its cognate sequence motifs have been detected in the genes of proteins implying its role in a variety of regulatory functions. The protein has a DNA binding domain consisting of three imperfect repeats with highly conserved tryptophans at regular spacings in each of the repeats. We have carried out a variety of investigations on the structure and interactions of the DNA binding domain of Drosophila c-myb and its cognate DNA target sequences. The domain has been bacterially over-expressed by subcloning a segment of the gene coding for the domain in a pET 11d vector and transforming it into E. coli BL21 (DE3). Circular dichroism of the protein has revealed that the domain is largely helical in nature. Fluorescence investigations indicated that three out of the nine tryptophans are solvent exposed and the others are buried in the interior. The recombinant protein is able to distinguish between specific and non-specific DNA targets in its binding and the interaction is largely electrostatic in nature in both cases. Dynamic fluorescence quenching experiments suggested that the DNA binding sites on the protein for specific and non-specific DNA targets are physically different. Most of the conserved tryptophans are associated with the specific DNA binding site. Simulated annealing and molecular dynamic simulations in a water matrix have been used to predict an energetically favoured conformation for the protein. Calculation of surface accessibilities of the individual residues shows that nearly 60% of the residues are less than 50% accessible to the solvent. Two and three dimensional NMR experiments with isotopically labelled protein have enabled spin system identification for many residue type and the types of residues involved in hydrophobic core formation in the protein. In an attempt to see the DNA surface possibly involved in specific interaction with the protein, a three-dimensional structure of a 12 mer cognate DNA has been determined by NMR in conjunction with restrained energy minimization. The recognition sequence shows interesting structural characteristics that may have important roles in specific interaction.

Amino Acid Sequence↗
PubMed1988

Transient expression in mammalian cells of the bacterial reporter gene encoding mercuric reductase: effects of various regulatory elements.

The effect of several transcriptional regulatory elements on gene expression in mammalian cells was investigated. As a reporter gene we have used the bacterial gene merA coding for the enzyme mercuric reductase. Several plasmids were constructed with different promoter/enhancer sequences (pSV/E, pSV/L, pMT, pRSV or pAd) at the 5' end and different splicing (small intron of the T antigen of SV40 or the second intron of the rabbit beta-globin gene) and/or polyadenylation signals (AEn, ALn or AR beta Gn) at the 3' end of the merA gene. Expression was measured in five different mammalian cell lines. In COS cells the highest level of expression is obtained with pSV/L and the lowest level with pSV/E. In HeLa, CV-1, Ltk-, and CHO cells merA expression is relatively high, under control of pRSV and pMT and relatively low under control of pSV/L and pAd. The introns studied have a negative effect on the expression of merA. The presence of a polyadenylation signal downstream from the gene is essential for its expression. The three different polyadenylation signals studied give a similar stimulatory effect on the level of expression of the merA gene.

Animals↗
PubMed2004

Nitration of a peptide phytotoxin by bacterial nitric oxide synthase.

Nitric oxide (NO) is a potent intercellular signal in mammals that mediates key aspects of blood pressure, hormone release, nerve transmission and the immune response of higher organisms. Proteins homologous to full-length mammalian nitric oxide synthases (NOSs) are found in lower multicellular organisms. Recently, genome sequencing has shown that some bacteria contain genes coding for truncated NOS proteins; this is consistent with reports of NOS-like activities in bacterial extracts. Biological functions for bacterial NOSs are unknown, but have been presumed to be analogous to their role in mammals. Here we describe a gene in the plant pathogen Streptomyces turgidiscabies that encodes a NOS homologue, and we reveal its role in nitrating a dipeptide phytotoxin required for plant pathogenicity. High similarity between bacterial NOSs indicates a general function in biosynthetic nitration; thus, bacterial NOSs constitute a new class of enzymes. Here we show that the primary function of Streptomyces NOS is radically different from that of mammalian NOS. Surprisingly, mammalian NO signalling and bacterial biosynthetic nitration share an evolutionary origin.

Arginine↗