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At least 1,189 records · Page 66Linked to original sources
PubMed1993

Escherichia coli host strains SURE and SRB fail to preserve a palindrome cloned in lambda phage: improved alternate host strains.

We have attempted to produce Escherichia coli strains with the optimal combination of host mutations required for the construction of genomic libraries in lambda and cosmid vectors. For lambda vectors, we defined this as a strain that combined high efficiency of phage plating with optimal tolerance to DNA methylation and the ability to propagate recombinants containing regions of potential secondary structure. To optimize this latter property, we have tested a series of strains for the ability to propagate a lambda phage containing a palindromic sequence. These included an mcr- derivative of a strain shown by Ishiura et al. [J. Bacteriol. 171 (1989) 1068-1074] to allow optimal stability of inserts in cosmid clones. All the sbcC strains allowed plaque formation of the palindrome-containing lambda phage. However, while the palindrome-containing phage plated with reasonable efficiency on SURE (recB sbcC recJ umuC uvrC) and SRB (sbcC recJ umuC uvrC), the majority of phage recovered from these strains no longer required an sbcC host for subsequent plating. These two strains also gave poorer titres with a low-yielding phage clone from the human Prader-Willi chromosome region. Optimal phage hosts appear to be those that are mcrA delta(mcrBC-hsd-mrr) combined with mutations in sbcC plus recBC or recD and without mutations in additional recombination functions such as recJ or recJ umuC uvrC (all of our E. coli strains are available on request).

Bacteriophage lambda↗
PubMed1996

A wound-inducible gene from Salix viminalis coding for a trypsin inhibitor.

A gene designated swin1.1 has been isolated by screening a Salix viminalis genomic library with a heterologous probe, win3 from Populus. The region sequenced included the entire coding sequence for a protein with 199 amino acids plus the promoter and terminator. At the 5' end of the coding region is a sequence that encodes a hydrophobic region of 25-30 amino acids, that could form a signal peptide. A putative TATAA box and polyadenylator sequence were identified. Introns were absent. The gene product showed similarities with serine protease inhibitors from the Kunitz family and especially with win3 from wounded leaves of Populus. Southern blot analysis indicated that swin1.1 is a member of a clustered gene family, swin1. An oligonucleotide corresponding to the putative hypervariable region towards the carboxyl end when used as a probe in Southern hybridization showed high specificity for swin1.1. Expression of the swin1.1 gene was enhanced in wounded leaves. The swin1.1 coding region without the signal sequence was highly expressed in Escherichia coli and the protein showed inhibitory activity against trypsin but at most slight activity against the other proteases tested. A systemically induced protein, SVTI, with inhibitor activity against trypsin, was isolated from Salix leaves by affinity chromatography on a column of trypsin-Sepharose 4B and N-terminal sequenced. It corresponded with the translated swin1.1 gene at 16 of the 19 amino acid sites, suggesting that SVTI is encoded by another member of the swin1 gene family.

Amino Acid Sequence↗
PubMed1995

Two Lactococcus lactis genes, including lacX, cooperate to trigger an SOS response in a recA-negative background.

A 4.3-kb EcoRI fragment from a Lactococcus lactis genomic library alleviates the methyl methanesulfonate, mitomycin C, and UV sensitivities of an Escherichia coli recA mutant (M. Novel, X. F. Huang, and G. Novel, FEMS Microbiol. Lett. 72:309-314, 1990). It complements recA1 and delta recA mutations but not recA13. Three proteins (with molecular masses of 20, 35, and 23 kDa) were produced from this fragment in a T7-directed system, and three corresponding genes were detected by DNA sequencing, namely, ISS1CH;lacX, which is the distal gene of the lac operon; and a third open reading frame, named lacN, which encodes 211 amino acids. Mutations produced in either lacX or in lacN resulted in the loss of the resistance to DNA-damaging agents. Thus, these two genes appeared to be involved in this activity. Introduction of pUCB214 carrying the 4.3-kb fragment into a lexA+ delta recA306 sfiA::lacZ strain resulted in UV-inducible synthesis of beta-galactosidase. A uvrA strain or a lexA (Ind-) strain containing pUCB214 did not support any DNA repair. However, a lexA (Def-) strain carrying pUCB214 could partly repair UV damage. We discuss possible targets for LacX and LacN products, and we speculate that LacX and LacN may constitute a two-component regulatory system that is able to respond to SOS signals, and then to act in the SOS response, bypassing the RecA-activated function.

Amino Acid Sequence↗
PubMed1994

A mutation in the indole-3-acetic acid biosynthesis pathway of Pseudomonas syringae pv. syringae affects growth in Phaseolus vulgaris and syringomycin production.

Homologs of the genes for indole-3-acetic acid (IAA) biosynthesis from Pseudomonas syringae pv. savastanoi were retrieved from a genomic library of P. syringae pv. syringae, and their nucleotide sequences were determined. Sequence relatedness between the P. syringae pv. syringae and P. syringae pv. savastanoi iaa operons is greater than 90% within the iaaM and iaaH loci but declines dramatically at a position approximately 200 bp 5' of the iaaM translation initiation codon. A third open reading frame was detected downstream of iaaH. Production of IAA was undetectable in mutant strain Y30-53.29, which was generated by transposition of Tn5 into the iaaM gene of P. syringae pv. syringae Y30. The IAA-deficient (IAA-) mutant retained the ability to colonize the bean phylloplane and induced disease symptoms on bean which were similar to those produced by the parental strain. However, the population dynamics of the IAA- strain during the parasitic phase in leaves differed from those of both the parental strain and the mutant genetically restored for IAA biosynthesis. The mutant was capable of inducing disease symptoms when established in bean tissues at a lower initial cell density than either IAA-producing strain. Syringomycin biosynthesis by the IAA- strain was diminished in comparison with the parental strain or the mutant genetically restored for IAA production. The results indicate that bacterially derived IAA, or its biosynthesis, is involved in the regulation of in planta growth and in the expression of other factors that affect the host-pathogen interaction.

Amino Acid Sequence↗
PubMed1997

The 75-kilodalton antigen of Bartonella bacilliformis is a structural homolog of the cell division protein FtsZ.

A genomic library of Bartonella bacilliformis was constructed and screened with human anti-Bartonella serum from a patient with the chronic, verruga peruana phase of bartonellosis. An immunoreactive clone isolated from this library was found to code for a 591-amino-acid protein with a high degree of sequence similarity to the FtsZ family of proteins. The degree of amino acid identity between the B. bacilliformis protein (FtsZ[Bb]) and the other FtsZ proteins is especially pronounced over the N-terminal 321 amino acids (N-terminal domain) of the sequence, with values ranging from 45% identity for the homolog from Micrococcus luteus (FtsZ[Ml]) to 91% identity for the homolog from Rhizobium melliloti, (FtsZ[Rm1]). All of the functional domains required for FtsZ activity are conserved in FtsZ(Bb) and are located within the N-terminal domain of the protein. FtsZ(Bb) is approximately twice as large as most of the other FtsZ proteins previously reported, a property it shares with FtsZ(Rm1). Like the Rhizobium homolog, FtsZ(Bb) has a C-terminal region of approximately 256 amino acids that is absent in the other FtsZ proteins. Evidence is presented that implicates this region in the protein's antigenicity and suggests that, unlike most other FtsZ homologs, FtsZ(Bb) is at least partly exposed at the cell surface. PCR analysis revealed that an ftsZ gene similar in size to the B. bacilliformis gene is present in Bartonella henselae, a bacterium that is closely related to B. bacilliformis.

Amino Acid Sequence↗
PubMed2001

The KlGpa1 gene encodes a G-protein alpha subunit that is a positive control element in the mating pathway of the budding yeast Kluyveromyces lactis.

The cloning of the gene encoding the KlGpa1p subunit was achieved by standard PCR techniques and by screening a Kluyveromyces lactis genomic library using the PCR product as a probe. The full-length open reading frame spans 1,344 nucleotides including the stop codon. The deduced primary structure of the protein (447 amino acid residues) strongly resembles that of Gpa1p, the G-protein alpha subunit from Saccharomyces cerevisiae involved in the mating pheromone response pathway. Nevertheless, unlike disruption of Gpa1 from S. cerevisiae, disruption of KlGpa1 rendered viable cells with a reduced capacity to mate. Expression of a plasmidic KlGpa1 copy in a DeltaKlgpa1 mutant restores full mating competence; hence we conclude that KlGpa1p plays a positive role in the mating pathway. Overexpression of the constitutive subunit KlGpa1p(K(364)) (GTP bound) does not induce constitutive mating; instead it partially blocks wild-type mating and is unable to reverse the sterile phenotype of DeltaKlgpa1 mutant cells. K. lactis expresses a second Galpha subunit, KlGpa2p, which is involved in regulating cyclic AMP levels upon glucose stimulation. This subunit does not rescue DeltaKlgpa1 cells from sterility; instead, overproduction of KlGpa2p slightly reduces the mating of wild-type cells, suggesting cross talk within the pheromone response pathway mediated by KlGpa1p and glucose metabolism mediated by KlGpa2p. The DeltaKlgpa1 DeltaKlgpa2 double mutant, although viable, showed the mating deficiency observed in the single DeltaKlgpa1 mutant.

Amino Acid Sequence↗
PubMed1991

Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line.

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.

Base Sequence↗
PubMed1993

Cloning of an endo-(1-->4)-beta-glucanase gene, celA, from the rumen bacterium Clostridium sp. ('C. longisporum') and characterization of its product, CelA, in Escherichia coli.

A genomic library of Clostridium sp. ('C. longisporum') ATCC 49440 in the host Escherichia coli was screened for endo-beta-glucanases, and plasmids pCM64 and pCM4 were isolated. The nucleotide sequence of a 3620 bp fragment was found to contain a 1548 bp open reading frame (ORF), termed celA, which encodes an endo-(1-->4)-beta-glucanase, CelA, assigned to family A4. N-terminal amino acid sequence determination revealed that pCM64 encoded the full-length celA gene, including a signal sequence, while pCM4 carried a 5'-truncated celA gene expressed as an N-terminal fusion protein, CelA delta N', without a signal sequence. CelA was secreted into the periplasm in E. coli. In this organism, proteolytic cleavage of CelA at or near a putative linker region resulted in the appearance of two active polypeptides of molecular masses 57 and 47 kDa. The former was the full-length enzyme while the latter consisted of the catalytic domain from which the cellulose-binding domain (CBD) had been removed (CelA delta CBD). The intracellularly-located CelA delta N' was not subject to proteolytic degradation. The pH and temperature optima of CelA were pH 4.8 and 43 degrees C, respectively. CelA hydrolysed barley beta-glucan, lichenan, carboxymethylcellulose and xylan. It showed preferential activity against the larger cellooligosaccharides (cellohexaose and cellopentaose); cellotetraose was the smallest substrate degraded completely.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and characterization of outer membrane protein E of Moraxella (Branhamella) catarrhalis.

Outer membrane protein E (OMP E) is a 50-kDa protein of Moraxella (Branhamella) catarrhalis. It is a potential vaccine antigen because it is expressed on the surface of the bacterium and has antigenic determinants which are conserved among most strains of M. catarrhalis. To clone the gene encoding OMP E, an EMBL-3 genomic library of strain 25240 was screened with a family of degenerate oligonucleotides based on the amino-terminal protein sequence. The OMP E gene was identified in one of the six positive clones by Southern blot analysis. An open reading frame of 1,377 bp encoding a protein of 460 amino acids was identified. The calculated molecular mass of the mature protein of 436 amino acid residues was 47.03 kDa, which correlated well with the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein product of the OMP E gene had a leader peptide of 25 amino acids and a signal peptidase 1 cleavage site similar to those of known OMPs of Escherichia coli. The transcription initiation site of the OMP E gene was mapped by primer extension to be 78 nucleotides upstream of the ATG start codon. Borderline homology was found to the FadL protein of E. coli (49.1% similarity and 25.6% identity), which is involved in the binding and transport of fatty acids. Analysis of restriction fragment length polymorphisms of the OMP E genes of 19 different strains of M. catarrhalis showed that the OMP E gene is highly conserved. The high degree of conservation of sequences of the OMP E genes of M. catarrhalis from diverse sources, along with earlier observations that the protein contains antigenic determinants on the bacterial surface, indicates that OMP E should be studied further as a potential vaccine antigen.

Amino Acid Sequence↗
PubMed1994

Human cathepsin S: chromosomal localization, gene structure, and tissue distribution.

The human lysosomal cysteine proteinases, cathepsins H, L, and B, have been mapped to chromosomes 15, 9, and 8, respectively, and the genomic structures of cathepsins L and B have been determined. We report here the chromosomal localization and partial gene structure for a recently sequenced human cysteine proteinase, cathepsin S. A 20-kilobase pair genomic clone of the human cathepsin S gene was isolated from a human fibroblast genomic library and used to map the human cathepsin S gene to chromosome 1q21 by fluorescence in situ hybridization. This clone contains exons 1 through 5, introns 1 through 4, part of intron 5, and > 7 kilobase pairs of the 5'-flanking sequence. The gene structure of human cathepsin S is similar to that of cathepsin L through the first 5 exons, except that cathepsin S introns are substantially larger. Sequencing of the 5'-flanking region revealed, similar to human cathepsin B, no classical TATA or CAAT box. In contrast to cathepsin B, cathepsin S contains only two SP1 and at least 18 AP1 binding sites that potentially could be involved in regulation of the gene. This 5'-flanking region also contains CA microsatellites. The presence of AP1 sites and CA microsatellites suggest that cathepsin S can be specifically regulated. Results of Northern blotting using probes for human cathepsins B, L, and S are consistent with this hypothesis; only cathepsin S shows a restricted tissue distribution, with highest levels in spleen, heart, and lung. In addition, immunostaining of lung tissue demonstrated detectable cathepsin S only in lung macrophages. The high level of expression in the spleen and in phagocytes suggests that cathepsin S may have a specific function in immunity, perhaps related to antigen processing.

Animals↗
PubMed2004

Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083.

Clones of a genomic library of Bifidobacterium adolescentis were grown in minimal medium with sucrose as sole carbon source. An enzymatic fructose dehydrogenase assay was used to identify sucrose-degrading enzymes. Plasmids were isolated from the positive colonies and sequence analysis revealed that two types of insert were present, which only differed with respect to their orientation in the plasmid. An open reading frame of 1,515 nucleotides with high homology for sucrose phosphorylases was detected on these inserts. The gene was designated SucP and encoded a protein of 56,189 Da. SucP was heterologously expressed in Escherichia coli, purified, and characterized. The molecular mass of SucP was 58 kDa, as estimated by SDS-PAGE, while 129 kDa was found with gel permeation, suggesting that the native enzyme was a dimer. The enzyme showed high activity towards sucrose and a lower extent towards alpha-glucose-1-phosphate. The transglucosylation properties were investigated using a broad range of monomeric sugars as acceptor substrate for the recombinant enzyme, while alpha-glucose-1-phosphate served as donor. D- and L-arabinose, D- and L-arabitol, and xylitol showed the highest production of transglucosylation products. The investigated disaccharides and trisaccharides were not suitable as acceptors. The structure of the transglucosylation product obtained with D-arabinose as acceptor was elucidated by NMR. The structure of the synthesized non-reducing dimer was alpha-Glcp(1-->1)beta-Araf.

Bifidobacterium↗
PubMed2003

Molecular cloning of the carboxylesterase gene and biochemical characterization of the encoded protein from Pseudomonas citronellolis ATCC 13674.

A genomic library of Pseudomonas citronellolis ATCC 13674 was constructed and screened for esterase activity in Escherichia coli using tributyrin-containing medium. One positive transformant was isolated, and subsequent analyses of the plasmid by restriction mapping revealed a 4.1-kb DNA fragment potentially carrying an esterase gene. The deduced nucleotide sequence of the DNA was found to contain an open reading frame encoding carboxylesterase and designated estA. Amino acid sequence analysis of estA showed the serine conservative motif, GDSAG, located between residues 208 and 212. Together with Ser, residues 310 and 334 corresponding to aspartic acid and histidine, respectively, comprised the catalytic triad. With the aid of immobilized metal ion affinity chromatography, the carboxylesterase fused with poly His at its C-terminus was purified and shown to be strongly inhibited by the tryptophan modifier and mercuric ion, indicating the important role of conservative Trp (189) and cysteine (152 and/or 183) residues in maintaining the structural integrity of the protein. Further analyses showed that the carboxylesterase functioned optimally at 37-40 degrees C with pH ranging between 8 and 9 and displayed a broad substrate spectrum. The protein exhibited greater preference toward short-chain (C2-C4) than medium- and long-chain fatty acids. Higher substrate specificity on para-nitrophenol butyrate was observed in comparison with para-nitrophenol acetate as indicated by the higher kcat/Km value of the former.

Amino Acid Sequence↗
PubMed1991

Latent a1 VH germline genes in an a2a2 rabbit. Evidence for gene conversion at both the germline and somatic levels.

We have previously reported the sequences of putative latent a1 cDNA derived from an alpha 2 alpha 2 rabbit. Significant similarity to nominal a1 cDNA sequences was noted, but none of the latent sequences were completely a1-like. We have now probed a genomic library, produced from the same alpha 2 alpha 2 rabbit, for evidence of germline latent a1 VH genes. Four hundred ninety-four VH+ clones were screened with oligonucleotides specific for a1 diagnostic regions of framework region 1 (FR1) and FR3. Twenty-two percent of the VH+ clones hybridized with an a1FR3 oligonucleotide probe. Two a1 FR1 probes yielded weak signals with 6% to 13% of the VH+ clones. Twenty VH genes from clones positive for one or more of the a1-specific oligonucleotide probes were sequenced, revealing 14 unique germline VH genes. All but one of these genes were 85% to 92% identical to the VH1-a1 nominal gene prototype, with sequence identity extending into the leader intron. Most genes displayed extended regions of similarity to a1 in FR1, FR3, or both and expressed 13 to 17 of the 21 allotype-associated residues, consistent with the nominal a1 sequence. The a1-like sequences were variously interspersed with short non-a1 segments, suggestive of germline gene conversion. Although none of the germline a1-like VH genes we have isolated from the alpha 2 alpha 2 rabbits are identical to the known a1 genes or protein sequences from alpha 1 alpha 1 rabbits and 8 of 14 are pseudogenes, most could make significant contributions to the synthesis of a complete nominal a1 sequence by serving as a pool of sequence donors during somatic gene conversion.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of two genes encoding proteases associated with the mycelium of Streptomyces lividans 66.

A strain of Streptomyces lividans 66 deleted for a major tripeptidyl aminopeptidase (Tap) was used as a host to screen an S. lividans genomic library for clones overexpressing activity against the chromogenic substrate Ala-Pro-Ala-beta-naphthylamide. In addition to reisolation of the tap gene, clones representing another locus, slpD, were uncovered. slpD was analyzed by deletion subcloning to localize its functional sequence. Nucleotide sequence determination revealed an open reading frame encoding a 55-kDa protein exhibiting significant amino acid sequence homology to Tap, particularly around the putative active-site serine residue. No secreted protein was observed for strains harboring the slpD clone, but inspection of the predicted protein sequence revealed a putative lipoprotein signal peptide (signal peptidase II type), suggesting a mycelial location for the SlpD proteinase. In an attempt to isolate an endoprotease known to be active against some heterologous proteins, a second clone was isolated by using a longer substrate (t-butyloxycarbonyl [Boc]-APARSPA-beta-naphthylamide) containing a chemical blocking group at the amino terminus to prevent aminopeptidase cleavage. This locus, slpE, appeared to also encode a 55-kDa mycelium-associated (lipoprotein) proteinase, whose predicted protein sequences showed significant amino acid homology to Tap and SlpD, particularly around the putative active-site serine residues. Chromosomal integration and deletion analysis in both the wild-type and Tap-deficient backgrounds appeared to indicate that SlpD was essential for viability and SlpE was required for growth on minimal media.

Amino Acid Sequence↗
PubMed1994

The exon structure of the mouse alpha 2(IX) collagen gene shows unexpected divergence from the chick gene.

One cosmid and two overlapping phage clones covering the entire mouse alpha 2(IX) collagen gene including 12 kilobase pairs (kb) of 5'- and 8 kb of 3'-flanking sequences were isolated from two genomic libraries. The overall gene structure was determined by restriction mapping and nucleotide sequencing. The gene spans 16 kb from the start of transcription to the polyadenylation site and contains 32 exons. It codes for a mRNA of 3 kb that translates into a polypeptide of 688 amino acids. The intron-exon junctions and mRNA structure were confirmed by amplification of cDNA made for mouse cartilage RNA. The coding sequence of the mouse alpha 2(IX) collagen gene shows marked similarities to those for other type IX collagen chains. Although the overall exon-intron organization of the mouse gene is very similar to the chick alpha 2(IX) gene, some unexpected differences were observed at the splice junctions. Split codons characteristic for the central triple helical domain of the chick were not found in the mouse gene that thus exhibited a long stretch of exons with sizes that are multiples of 9 base pairs in this domain. The promoter of the mouse alpha 2(IX) collagen gene contains some G + C-rich elements including three Sp1 consensus recognition sites and a far upstream CCAAT box but no TATAA box. Both primer extension and RNase protection assays revealed several transcription start sites within 418 base pairs of the promoter. The present study reports the first complete nucleotide sequence of any type IX collagen gene and forms the basis for comparative structural studies on this collagen type and for experiments involving transgenic mice.

Amino Acid Sequence↗
PubMed1999

Characterization of an atypical superoxide dismutase from Sinorhizobium meliloti.

Sinorhizobium meliloti Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a sod fragment amplified by PCR from degenerate primers as a probe. The sodA gene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical M(r) of 22,430 and pI of 5. 8. S. meliloti SOD complemented a deficient E. coli mutant, restoring aerobic growth of a sodA sodB recA strain, when the gene was expressed from the synthetic tac promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H(2)O(2). The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer). The apoenzyme was prepared from crude extracts of S. meliloti. Activity was restored by dialysis against either MnCl(2) or Fe(NH(4))(2)(SO(4))(2), demonstrating the cambialistic nature of the S. meliloti SOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H(2)O(2). Sequence comparison with 70 FeSODs and MnSODs indicates that S. meliloti SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H(2)O(2) of this unusual Fe-type SOD.

Amino Acid Sequence↗
PubMed1995

Cloning, sequence analysis, and expression of the gene encoding formaldehyde dismutase from Pseudomonas putida F61.

The gene (fdm) coding for formaldehyde dismutase (EC 1.2.99.4) from a genomic library of formaldehyde-tolerant Pseudomonas putida F61 was cloned and expressed in Escherichia coli. The nucleotides of the cloned DNA were sequenced; they included a single open reading frame of 1200 base pairs, coding for a putative protein with a molecular weight of 42,848. Sequencing of the first 20 N-terminal amino acid residues and of an internal part of the enzyme purified from P. putida F61 established the identity and the start codon of fdm. Comparison of the amino acid sequence predicted from fdm with that of alcohol dehydrogenase from horse liver suggested a putative pyridine-dinucleotide-binding domain in fdm, and also potential ligands for the catalytic domain and the second zinc atom-folding domain. fdm seemed to be expressed in E. coli under control of the promoter of fdm; there was an E. coli promoter-like sequence upstream from the gene. The enzyme expressed in E. coli was purified to homogeneity. The molecular weight and the sequence of the first 20 N-terminal amino acid residues were identical with those of P. putida formaldehyde dismutase. Each subunit contained 1 mol of NAD(H) and 2 mol of zinc per mol of protein. The enzyme produced in E. coli catalyzed the dismutation of formaldehyde to form methanol and formic acid at the ratio of 1:1 in the absence of the exogenous electron acceptor, NAD(H).

Alcohol Dehydrogenase↗
PubMed1992

Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters.

The expression of the beta isoenzyme for protein kinase C is regulated developmentally and in response to inducers of cell differentiation (such as phorbol esters and 1 alpha,25-dihydroxyvitamin D3). The 5' segment of the gene for protein kinase C beta was cloned from a human leukocyte genomic library in EMBL3 bacteriophage. This segment of the gene (greater than 54 kilobases in length) encompassed the coding sequence for the amino-terminal regulatory domain of the enzyme, the 5'-untranslated region, and the 5'-flanking region. Initiation of transcription was identified by S1 nuclease analysis and confirmed by RNase protection analysis at 197 base pairs 5' of the initiator ATG. Sequence analysis of the 5'-flanking region revealed it to be extremely G+C-rich (> 80%) with many features of a CpG island. Comparison of sequence with known cis-regulatory motifs disclosed a number of potential regulatory elements including an octamer binding motif at -76, Sp1-binding sites at -94 and -63, E boxes at -110, -26, and +18, an AP-1 site at -442, and an AP-2 site at -330. To demonstrate promoter activity, a 630-base pair fragment extending from -587 to +43 was subcloned in front of a promoterless luciferase gene. This fragment was able to drive the expression of luciferase in transient transfections of human hematopoietic cells. Deletion analysis demonstrated that a fragment -111 to +43 was necessary and sufficient for promoter activity; this fragment did not contain TATA or CAAT motifs. The promoter was stimulated 8-20-fold by phorbol esters accounting for the previously observed transcriptional activation of protein kinase C beta. This phorbol ester responsiveness was conferred by the basal promoter (-111 to +43) and was independent of the AP-1 site. These results define a novel mechanism of protein kinase C autoregulation at a transcriptional level.

Animals↗