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.     

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, sequencing, characterization, and expression of a new α-amylase isozyme gene (amy3) from Pseudomonas sp.

A new gene encoding an -amylase has been cloned, sequenced and expressed in E. coli from an alkaliphilic Pseudomonas sp. KFCC10818. The structural gene is 1356 base pairs long and encodes a protein of 452 amino acids. The recombinant -amylase has been purified and biochemically characterized. Molecular mass of the protein deduced from SDS-PAGE was 50 kDa. The enzyme showed an activity optimum at pH 8 and at 40 °C with complete stability at pH 13 for 3 h. The enzyme released maltose and maltotriose on hydrolysis of soluble starch. Amylose was hydrolysed over 5 times faster than amylopectin by the enzyme while the hydrolysis of cyclodextrin or pullulan was negligible.     

Cloning, sequencing, and heterologous expression of an Erwinia cypripedii 314B lactonase specific for l-α-hydroxyglutaric acid γ-lactone

The gene for a lactonase that stereospecifically hydrolyzes (S)-5-oxo-2-tetrahydrofurancarboxylic acid to l-α-hydroxyglutaric acid was isolated from Erwinia cypripedii 314B. Determination of the nucleotide sequence showed that the gene consists of a single open reading frame of 1,152 bp that encodes a 383-amino-acid protein. Comparison of the sequence of the predicted protein to that of the enzyme purified from E. cypripedii 314B revealed an N-terminal signal sequence of 19 amino acids. The gene for the mature enzyme was inserted into a pET vector and overexpressed in Escherichia coli. Active recombinant enzyme accumulated in the cells to ∼30% of the total protein, and the enzyme was purified to homogeneity. The physical and catalytic properties of the recombinant enzyme were indistinguishable from those of the protein purified from E. cypripedii 314B. The deduced amino acid sequence displayed ∼35% similarity with a putative 3-carboxymuconate cyclase, but exhibited no such activity. The enzyme also showed ∼35% similarity with 6-phosphogluconolactonase. However, the activity of the enzyme toward 6-phosphogluconolactone was less than 2% of that toward (S)-5-oxo-2-tetrahydrofurancarboxylic acid, demonstrating a novel specificity for this lactonase.     

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 of a Picea abies monosaccharide transporter gene and expression – analysis in plant tissues and ectomycorrhizas

Ectomycorrhizas are formed between certain soil fungi and fine roots of woody plants. An important feature of this symbiosis is the supply of photoassimilates to the fungus. Hexoses, formed from sucrose in the common apoplast at the root/fungus interface, can be taken up by both plant and fungal monosaccharide transporters. Recently we characterised a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. This transporter was up-regulated in mycorrhizas, thus increasing the hexose uptake capacity of the fungal partner in symbiosis. In order to characterise host (Picea abies) root monosaccharide transporters, degenerate oligonucleotide primers, designed to match conserved regions from known plant hexose transporters, were used to isolate a cDNA fragment of a transporter by PCR. This fragment was used to identify a presumably full length clone (PaMST1) in a P. abies/A. muscaria mycorrhizal cDNA library. The entire cDNA code for an open reading frame of 513 amino acids, revealing best homology to H⁺/monosaccharide transporters from Ara- bidopsis, Saccharum and Ricinus. PaMST1 was highly expressed in the hypocotyl and in roots of P. abies seedlings, but not in needles. Mycorrhiza formation led to a slight reduction of PaMST1 expression. The results are discussed with special reference to carbon allocation in ectomycorrhizas. Received: 9 October 1999 / Accepted: 22 December 1999     

Purification and partial characterization of ad(−)-lactate dehydrogenase fromDesulfovibrio desulfuricans (ATCC 7757)

Summary A membrane-boundd(–)-lactate dehydrogenase (LDH), an important enzyme in carbon and energy metabolism in sulfate-reducing bacteria of the genusDesulfovibrio, was solubilized from the membrane fraction ofDesulfovibrio desulfuricans (ATCC 7757). The enzyme was purified 84-fold to a final specific activity of 525 nmol DCPIP-reduced/min/mg protein by ammonium sulfate precipitation, chloroform extraction, gel filtration with Sephadex G-150, and hydrophobic column chromatography withN-octylamine Sepharose 4B. The enzyme eluted off a Sephacryl S-300 column as a single peak with a molecular weight of 400 000±40 000 Da. Denaturing gel electrophoresis showed it to be composed of 5 protein bands. The oxidized and dithionite reduced spectra of LDH resembles the spectra ofc-type cytochromes found inDesulfovibrio species. The addition of lactate to LDH resulted in a partially reduced spectrum. The flavin/cytochromec/non-heme iron content per 400 000 Da LDH molecular weight was found to be 11.64.5. The LDH activity was specific ford(–)-lactate and had aK _m ford(–)-lactate of 4.3×10^–4 M. The pH optimum was between 6.5 and 8.5.     

Isolation and characterization of two sorbitol transporter gene promoters in micropropagated apple plants (Malus?×?domestica) regulated by drought stress

The role of polyol transporters in stress tolerance in plants have been elucidated by many studies. Sorbitol transporter genes MdSOT3, MdSOT4 and MdSOT5 in apple plants, which are important for sorbitol loading and unloading, are regulated by drought stress. To further confirm the role of sorbitol transporters in stress tolerance, the constructs harboring MdSOT3 and MdSOT5 genes were introduced into wild type Arabidopsis plants (Col-0) and the Arabidopsis transformed with MdSOT3 or MdSOT5 performed higher drought stress tolerance compared to WT. In order to further understand how sorbitol transporters are involved in drought tolerance in apple plants, upstream regions of sorbitol transporter genes were isolated from apple plant source leaves by Anchored PCR from genomic DNA obtained, and then were used to drive expression of the GUS reporter in tobacco plants. The results showed that the longest fragments of MdSOT3 and MdSOT5 promoters induced the highest GUS activity under drought stress conditions. Additionally, fragments of these promoters that contain cis-acting elements known to be involved in stress response also induced GUS activity under drought stress. Taken together, our data suggest that increased MdSOT3 and MdSOT5 activity, through cis-acting elements in the promoters of these genes, play important roles in imparting tolerance to drought in micropropagated apple plants.     

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,characterisation and expression of the α-tubulin genes of the leech,Hirudo medicinalis

We have isolated two α-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two α-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo α-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech α-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod α-tubulins. The protein sequences of the leech α-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.     

Cloning and nucleotide sequence of the β-galactosidase gene from Lactococcus lactis ssp. Lactis ATCC7962

The gene encoding -galactosidase of Lactococcus lactis ssp. lactis ATCC7962 was cloned and its nucleotide sequence was determined. The -galactosidase of L. lactis was expressed in Escherichia coli and transformants containing this gene fragment appeared as blue colonies on LB plates containing X-gal. The -galactosidase activity of E. coli transformant was thirty times higher than that of L. lactis. The gene for the 115 kDa -galactosidase has a 2991-bp open reading frame preceded by a putative ribosome binding site. The deduced amino acid sequence show a high degree of homology to the -galactosidase of E. coli, and the putative active site residues are conserved (Glu-429 and Tyr-475)     

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 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 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.