Cloning and sequencing of the hemA gene of Rhodobacter capsulatus and isolation of a δ-aminolevulinic acid-dependent mutant strain

Summary The Rhodobacter capsulatus hemA gene, coding for the enzyme -aminolevulinic acid synthase (ALAS), was isolated from a genome bank by hybridization with a hemT probe from Rhodobacter sphaeroides. Subcloning of the initial 3.9 kb HindIII fragment allowed the isolation of a 2.5 kb HindIII-BglII fragment which was able to complement the -aminolevulinic acid-requiring (ALA-requiring) Escherichia coli mutant SHSP19. DNA sequencing revealed an open reading frame coding for a protein with 401 amino acids which displayed similarity to the amino acid sequences of other known ALASs. However, no resemblance was seen to the HemA protein of E. coli K12. Based on the sequence data, an ALA-requiring mutant strain of R. capsulatus was constructed by site-directed insertion mutagenesis. Introduction of a plasmid, containing the hemA gene of R. capsulatus on the 3.9 kb HindIII fragment, restored ALA-independent growth of the mutant indicating that there is only one gene for ALA biosynthesis in R. capsulatus. Transfer of the R factor pRPS404 and hybridization analysis revealed that the ALAS gene is not located within the major photosynthetic gene cluster.Part of this research was presented at the Symposium on Molecular Biology of Membrane-Bound Complexes in Phototrophic Bacteria, Freiburg, FRG, 2–5 August 1989     

Cloning,partial sequencing and 17β-estradiol modulation of hepatic vitellogenin gene of the Neotropical catfish Rhamdia quelen

Expression of vitellogenin gene (vtg) is used as a biomarker for the evaluation of the exposure of estrogenic substances in fish. However, scientific information regarding this biomarker in Neotropical fish is limited. In this study, a 760 bp partial sequence of the vtg mRNA from the liver of the catfish Rhamdia quelen was cloned, representing almost 20% of the full-length vtg. The phylogenetic tree analysis recovered the vtg of R. quelen inside the clade of Siluriformes. The alignment of the deduced amino acid sequence of R. quelen vtg with other species confirmed that the described sequence is gene specific. Also, this cloned sequence presents almost 70% of identity with the vtgI subtype, that is known to translate into the incomplete form of Vtg due to the lack of the two domains, the β’-c and the Ct. Moreover, from the cloning and sequencing of vtg of R. quelen, a protocol for a RT-qPCR was developed with the goal to be applied as a biomarker of the hypothalamic-pituitary-gonad-liver (HPGL) axis, in order to evaluate the effects of endocrine disruptor exposure in Neotropical fish. Thus, this protocol was applied in the effects of a dose of 10 mg/kg of 17β-estradiol (E2) on vtg expression in male and female fish. The results showed that after 17 days of the exposure injection the E2 treatment upregulated vtg expression in both sexes. No observed differences in the levels of gene expression between the male and female fish were observed, and no statistical interaction between E2-treatment and sex were found. The results obtained from cloning add new information regarding vtg in Siluriformes fish, an order poorly studied in this aspect. Also, the vtg RT-qPCR protocol stablished for vtg of R. quelen will expand the application of this animal model, a Neotropical fish, in investigation regarding the effects of endocrine disruptors.     

Interaction of measles virus (Hallé strain) with CD46: evidence that a common binding site on CD46 facilitates both CD46 downregulation and MV infection.

CD46 acts as a cellular receptor for vaccine strains of measles virus (MV). The MV/CD46 interaction-mediated by the MV attachment glycoprotein, the hemagglutinin (H)-not only facilitates infection but also induces CD46 downregulation. A conflict of opinion exists as to whether a single MVH binding site on CD46, or two separate sites, facilitates the two phenomena. To investigate this conundrum we first tested and compared a panel of CD46-specific monoclonal antibodies (mAbs) for their capacity to block both processes. One (mAb 13/42) abrogated both MV fusion and CD46 downregulation. Mutation of an amino acid (arg59 in the SCR1 of CD46) essential for the epitope of mAb 13/42 resulted in the abrogation of both CD46 downregulation and viral fusion. This strongly suggests that the same MV binding site on CD46 is responsible for both CD46 downregulation and MV infection.     

Cloning and sequencing of a carp βs-crystallin cDNA

The mRNAs were extracted from common carp (Cyprinus carpio) lenses, purified, reverse transcribed, dC tailed and cloned into Escherichia coli with pBR322 as vector. The cloning efficiency was around 1·10⁷ colonies per μg of mRNA. A clone (pC20) was found by hybrid-arrested translation to contain the cDNA related to carp crystallins. However, comparison of the derived amino-acid sequence with bovine γ-II and β_s-crystallins indicates that this carp crystallin sequence resembles closely the bovine β_s-crystallin and should be better classified as such except that this fish sequence does not contain the N-terminal ‘arm’ of four amino-acid residues present in bovine β_s-crystallin.     

Cloning,sequencing and analysis of the ggh-A gene encoding a 1,4-β-d-glucan glucohydrolase from Microbispora bispora

The ggh-A gene, encoding a 1,4-β-d-glucan glucohydrolase/β-glucosidase, of Microbispora bispora (Mb) was subcloned and expressed from a 4.0-kb XhoI DNA fragment. The nucleotide sequence of this fragment was determined. Analysis of the sequence revealed one open reading frame (ORF) which encodes a 986-amino-acid (aa) protein with a calculated molecular weight of 107 510. The ggh-A ORF has features typical of an actinomycete gene including high GC content (70.5%) and corresponding biased codon usage. Comparison of the aa sequence of the Mb 1,4-β-d-glucan glucohydrolase (Mbggh-A) with other glycosidases reveals high overall homology to several β-glucosidases and a 1,4-β-d-glucan glucohydrolase belonging to the glycosyl hydrolase family 3. The aa sequence alignments of Mbggh-A and β-glucosidases show that the active site region potentially involves two Asp residues. The aa sequence homology studies revealed a potential two-domain structure for Mbggh-A and other β-glucosidases. Furthermore, Mbggh-A has localized homology to a cellulose-binding domain present in some xylanases. This report is significant, as, to date, 1,4-β-d-glucan glucohydrolases have rarely been reported, though they are assumed to have a critical role in cellulolysis.     

Cloning, sequencing, and expression of a β-1,4-endoxylanase gene from Indonesian Bacillus licheniformis strain I5 in Escherichia coli

The gene encoding an endo-β-1,4-xylanase from an Indonesian indigenous Bacillus licheniformis strain I5 was amplified using PCR, cloned, and expressed in Escherichia coli. The nucleotide sequence of a 642 bp DNA fragment was determined, revealing one open reading frame that encoded a xylanase. Based on the nucleotide sequence, calculated molecular mass of the enzyme was 23 kDa. This xylanase has a predicted typical putative signal peptide; however, in E. coli, the active protein was located mainly in intracellular form. Neither culture supernatant of recombinant E. coli nor periplasmic fraction has significantly detectable xylanase activity. The deduced amino acid of the gene has 91% identity with that of Bacillus subtilis endoxylanase. Optimal activity of the recombinant enzyme was at pH 7 and 50°C     

Cloning,sequencing and partial characterisation of sorbitol transporter (srlT) gene encoding phosphotransferase system,glucitol/sorbitol-specific IIBC components of Erwinia herbicola ATCC 21998 – Cloning,sequencing and partial characterisation of sorbitol specific transporter (srlT) gene of Erwinia herbicola ATCC 21998

A DNA fragment of approximately 1500 bp, harbouring the sorbitol transport gene (srlT), was amplified from the chromosomal DNA of Erwinia herbicola ATCC 21998 by PCR and cloned in Escherichia coli JM109. Degenerate oligonucleotide primers used were designed based on the conserved regions in the gene sequences within the gut operon of E. coli (Gene Bank accession no. J02708) and the srl operon of Erwinia amylovora (Gene Bank accession no. Y14603). The cloned DNA fragment was sequenced and found to contain an open reading frame of 1473 nucleotides coding for a protein of 491 amino acids, corresponding to a mass of 52410 Da. The nucleotide sequence of this ORF was highly homologous to that of the gutA gene of Escherichia coli gut operon, the srlE gene of Shigella flexrni and the sorbitol transporter gene sequence of Escherichia coli K12 (Gene Bank accession nos. J02708, AE016987 and D90892 respectively). The protein sequence showed significant homology to that of the phosphotransferase system i.e. the glucitol/sorbitol-specific IIBC components of Escherichia coli and Erwinia amylovora (P56580, O32522). The cloned DNA fragment was introduced into a pRA90 vector and the recombinant was used for developing srlT mutants of Erwinia herbicola, by homologous recombination. Mutants obtained were unable to grow on minimal medium with sorbitol. The insertion of the pRA90 vector inside the srlT gene sequence of the mutants was confirmed by DNA-DNA hybridisation.     

Cloning and expression of the 3′ portion of the T4 denV gene as a lacZ fusion gene

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage λgt11 T4 dC DNA library was screened for phage which produced a β-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25 % of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within λgt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native β-galactosidase.     

Cloning and sequencing of the equine and ovine high-affinity IgE receptor β-and γ-chain cDNA

The high-affinity receptor for IgE is expressed on the surface of mast cells and basophils. It is a transmembrane protein with one alpha, one beta and two gamma subunits. The cDNA sequences for the alpha subunit have already been determined. We report here the cDNA sequences for the beta and gamma subunits. The cytoplasmic domains of these subunits are important for intracellular signalling and the deduced amino acid sequences show the expected immunoreceptor tyrosine-based activation motifs. The gamma subunit is highly conserved between species but more variation is seen with the beta subunit. Near the C terminus of the equine beta chain there is a two-base deletion, which changes the reading frame: residue 237 (human numbering) becomes Asp instead of Glu and the chain is three amino acids shorter than the other known mammalian and rodent sequences.     

Cloning and sequencing of a novel glutaryl acylase β-subunit gene ofPseudomonas cepacia BY21 from bioinformatics

Pseudomonas cepacia BY21 was found to produce glutaryl acylase that is capable of deacylating glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) to 7-aminocephalosporanic acid (7-ACA), which is a starting material for semi-synthetic cephalosporin antibiotics. Amino acids of the reported glutaryl acylases from variousPseudomonas sp. strains show a high similarity (>93% identity). Thus, with the known nucleotide sequences ofPseudomonas glutaryl acylases in GenBank, PCR primers were designed to clone a glutaryl acylase gene fromP. cepacia BY21. The unknown β-subunit gene of glutaryl acylase from chromosomal DNA ofP. cepacia BY21 was cloned successfully by PCR. The β-subunit amino acids ofP. cepacia BY21 acylase (GenBank accession number AY948547) were similar to those ofPseudomonas diminuta KAC-1 acylase except that Asn408 ofP. diminuta KAC-1 acylase was changed to Leu408.     

Cloning of the β2-microglobulin gene in the zebrafish

The ₂-microglobulin (₂m) is a protein found in the serum in a free form and on the cell surface in a form noncovalently associated with the chain of the class I major histocompatibility complex (Mhc) molecules. In mammals, the ₂m-encoding gene (B2m) is found on a chromosome different from the Mhc proper. We have isolated and characterized the B2m gene of the zebrafish, Brachydanio rerio, family Cyprinidae. We obtained both cDNA and genomic clones of the Brre-B2m gene. The cDNA clones contained the entire coding sequence, the entire 3 untranslated (UT) region, and at least part of the 5UT region. The genomic clone contained the entire Brre-B2m gene. The coding sequence specifies 97 amino acid residues of the mature protein so that the zebrafish ₂m is two residues shorter than human and one residue shorter than cattle, fowl, or turkey ₂m (codons at positions 85 and 86 have been deleted in the Brre-B2m. gene). The amino acid and nucleotide sequence similarities between zebrafish and human ₂m (B2m) are 45% and 59%, respectively. Approximately 24% of the positions are invariant and an additional 9% show only conservative substitutions in comparisons which include all known 2m sequences (fish, avian, and mammalian). Most of the conserved positions are in the strands (some 47% of the -strand positions are conserved in the three vertebrate classes). The Brre-B2m gene consists of four exons separated by three introns. All of the introns are considerably shorter than the corresponding introns in the mammalian B2m genes. The coding sequences of the cDNA and the genomic clones are almost identical but the sequences of the 3'UT regions differ at 1.7% of the sites, suggesting that the genes borne by these clones might have diverged at least 0.7 million years (my) ago. In contrast to the human B2m gene, the Brre-B2m gene shows no bias in the distribution of the CpG dinucleotides: the dinucleotides are distributed evenly along the entire available sequence. The haploid genome of the zebrafish contains only one copy of the B2m gene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L05383 (B2M) and L05384 (B2RG). Correspondence to: J. Klein.     

Cloning sequencing and expression of the gene for cytochrome P450meg,the steroid-15β-monooxygenase from Bacillus megaterium ATCC 13368

A 4.3 kb EcoRI fragment carrying the gene for cytochrome P450meg, the steroid-15β-monooxygenase from Bacillus megaterium ATCC 13368, was cloned and completely sequenced. The gene codes for a protein of 410 amino acids and was expressed in Escherichia coli and B. subtilis. Protein extracts from the recombinant E. coli strains were able to hydroxylate corticosteroids in the 15β position when supplemented with an extract from a P450- mutant of B. megaterium ATCC 13368 as a source of megaredoxin and megaredoxin reductase. In contrast, 15β-hydroxylation was obtained in vitro and in vivo without the addition of external electron transfer proteins, when cytochrome P450meg was produced in B. subtilis 168. Protein extracts from nonrecombinant B. subtilis 168 could also support the in vitro hydroxylation by cytochrome P450meg produced in E. coli.     

Cloning and expression of the α-amylase gene and oxytetracycline production in Streptomyces rimosus

An 8.4 kb Sau3AI DNA fragment containing the Streptomyces rimosus TM-55 -amylase gene (amy) was ligated to a vector pIJ702, named pCYL01, and cloned into amylase deficient mutant S. lividans M2 (amy ^–). Subcloning study showed that the amy gene was localized in 3.3 kbKpnI-PstI fragment. The molecular weight of the purified -amylases of S. lividans M2/pCYL01 and S. rimosus TM-55 were estimated to be 65.7 kDa. Different sizes of recombinant plasmids carrying the amy gene had been retransferred into the parental strain of S. rimosus TM-55. Among these S. rimosus transformants, TM-55/pCYL01, TM-55/pCYL12 and TM-55/pCYL36 showed amylase activity 1.36- to 2.05-fold at the seventh day (1.61 to 2.42 units vs 1.18 units), and oxytetracycline (OTC) production 2.00- to 2.50-fold at the ninth day (approximate 140 to 170 g ml^–1 vs 72 g ml^–1), higher than that of S. rimosus TM-55 alone, respectively. These results showed that industrial microorganisms could be improved by genetic and metabolic engineering.     

Cloning and expression of a chicken α-amylase gene

We have isolated and sequenced a genomic clone for a pancreatic α-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY) of 512 aa that is 83% identical to the human pancreatic α-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the α-amylase produced by the yeast cells was identical to that of the native chicken enzyme.     

Is mRNA and protein level of CD46 altered in measles virus vaccine strain S191-infected cells?

Previous research showed that the expression of measles virus receptor CD46 was downregulated after expression of measles virus hemagglutinin protein on the surface of the virus infected cell or triggered by infected cell-to-cell contact. We reported here that the mRNA level of CD46 in MV infected cells was not changed which was tested by real-time quantitative PCR. To further analyse the surface expression alteration of CD46 after MV infection, flow cytometric analysis and indirect immunofluorescence were used to detect the protein level of CD46. Altogether, our results provided a demonstration that the expression of CD46 was not downregulated by the infection of MV strain S191 both on mRNA level and cellular surface protein level. Previous results reported that the "downregulation" of CD46 expression on the cell surface may take place because H protein masks the antibody recognition site on CD46 which results in "downregulation" of the expression of CD46.     

Cloning and functional analysis of the Gβ gene Mgb1 and the Gγ gene Mgg1 in Monascus ruber

The ascomycetous fungus Monascus ruber is one of the most well-known species widely used to produce Monascus-fermentation products for natural food colorants and medicine. Our previous research on the Gα subunit Mga1 and the regulator of G protein signaling MrflbA indicated that heterotrimeric G protein signaling pathways were involved in aspects of growth, sporulation and secondary metabolite production in M. ruber. To better understand the G protein signaling pathways in this fungus, a Gβ subunit gene (Mgb1) and a GΓ subunit gene (Mgg1) were cloned and investigated in the current study. The predicted Mgb1 protein consisted of 353 amino acids and Mgg1 consisted of 94 amino acids, sharing marked similarity with Aspergillus Gβ and GΓ subunits, respectively. Targeted deletion (Δ) of Mgb1 or Mgg1 resulted in phenotypic alterations similar to those resulting from ΔMga1, i.e., restricted vegetative growth, lowered asexual sporulation, impaired cleistothecial formation, and enhanced citrinin and pigment production. Moreover, deletion of Mgg1 suppressed the defects in asexual development and in biosynthesis of citrinin and pigment caused by the absence of MrflbA function. These results provide evidence that Mgb1 and Mgg1 form a functional GβΓ dimer and the dimer interacts with Mga1 to mediate signaling pathways, which are negatively controlled by MrflbA, for growth, reproduction and citrinin and pigment biosynthesis in M. ruber.     

Cloning and expression of the α–L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae

 First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1 _P ) and terminator (PGK1 _T ) sequences on a multicopy episomal plasmid. The resulting construct PGK1 _P -abf B-PGK1 _T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively. Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996