Evolution of single-copy DNA and the ADH gene in seven drosophilids

Summary Single-copy DNA was isolated fromDrosophila melanogaster and hybridized with total genomic DNA ofD. melanogaster, D. mauritiana, D. simulans, D. pseudoobscura, D. willistoni, D. hydei andD. virilis. The duplexes were thermally eluted from hydroxyapatite and the data used to assess the relatedness of each species toD. melanogaster. The general pattern of relatedness was similar to that predicted by morphological methods but with some notable exceptions. The rate of nucleotide substitution was estimated to be greater than 0.66% of bases per million years. An unexpected, rapidly evolving component ofD. melanogaster single-copy DNA was identified. The relatedness of these species was also studied with respect to the gene coding for alcohol dehydrogenase (ADH). The ADH gene, previously cloned fromD. melanogaster (Goldberg 1980), was hybridized with Southern blots of genomic digests of the seven species. The intensity and position of the hybridizing bands suggest the amount of divergence of the gene. Divergence was quantitated by reassociation of a fragment of the cloned ADH gene with total DNA of the seven drosophilids and thermal elution of the resultant duplexes from hydroxyapatite. The ADH gene was isolated from genomic clone libraries ofD. melanogaster, D. simulans andD. mauritiana and further studied by comparison of position of restriction sites. Species relationships deduced from comparison of total single-copy DNA and the ADH gene were consistent, demonstrating that a single gene can reflect divergence of the entire genome.     

Isolation and Identification of Novel Terpene Lactones from Quince Fruit (Cydonia oblonga Mill., Marmelo)

A glucoamylase gene has been cloned from a Rhizopus genomic DNA library using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a cDNA library prepared from Rhizopus mRNA. Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the cDNA gene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65,000. The glucoamylase gene to be expressed in yeast cells was constructed by recombination of both genes. The yeast cells containing this constructed glucoamylase gene secreted the glucoamylase into the culture fluid and grew at almost the normal rate on a medium containing starch as the sole carbon source.     

Cloning and characterization of genes coding for ribosomal RNA inCampylobacter jejuni

The DNA fragments coding for ribosomal RNA inCampylobacter jejuni have been cloned from a genomic library ofC. jejuni constructed inEscherichia coli. Clones carrying DNA Sequences for rRNA were identified by hybridization of 5-end-labeled rRNA fromC. jejuni to colony blots of transformants from this gene library. Cloned DNA sequences homologous to each of 5S, 16S, and 23S rRNA were idenfified by hybridization of labeled plasmid DNA to Northern blots of rRNA. The gene coding for 23S rRNA was found to be located on a 5.5kb HindIII fragment, while the 5S and 16S rRNA genes were on HindIII fragments of 1.65 and 1.7 kb, respecitively. The DNA fragment containing the 16S rRNA gene was characterized by restriction endonuclease mapping, and the location of the 16S rRNA gene on this fragment was determined by hybridization of 5-end-labeled rRNA to restriction fragments and also by DNA sequence determination. It appears that the major portion of the coding region for 16S rRNA is located on the 1.7-kb HindIII fragment, while a small portion is carried on an adjacent HindIII fragment of 7.5 kb. Cloned rRNA genes fromC. jejuni were used to study the organization of the rDNA inC. jejuni and other members of the genùsCampylobacter.     

Relationships between species ofLeymus,Psathyrostachys, andHordeum (Poaceae,Triticeae) inferred from Southern hybridization of genomic and cloned DNA probes

We have used total genomic DNA as a probe to size-fractionated restriction enzyme digests of genomic DNA from a range ofTriticeae species from the generaLeymus Hochst.,Psathyrostachys Nevski, andHordeum L., and hybrids betweenHordeum andLeymus to investigate their taxonomic relationships. Genomic Southern hybridization was found to be an effective and simple way to assess the distribution and diversity of essentially species-specific and common, repetitive DNA sequences, and is hence especially useful in evolutionary studies. The DNA sequences ofH. vulgare seem to diverge substantially from those ofH. brachyantherum, H. lechleri, H. procerum, andH. depressum. The genome ofThinopyron bessarabicum shows little homology to those of theLeymus species investigated, confirming thatT. bessarabicum is not an ancestral genome inLeymus. Although the genomes ofLeymus andPsathyrostachys share substantial proportions of DNA sequences, they include divergent repeated sequences as well. Hybridization with a ribosomal DNA probe (pTa 71) showed that the coding regions containing structural genes encoding the 18 S, 5.8 S, and 26 S ribosomal RNA were conserved among the species investigated, whereas the intergenic spacer region was more variable, presenting different sizes of restriction fragments and enabling a classification of the species. The rye heterochromatin probe pSc 119.2 hybridized to DNA fromH. lechleri andT. bessarabicum, but not to DNA from the other species investigated.     

Relationships betweenOryza species (Poaceae) based on 5S DNA sequences

Relationships between 9Oryza species, covering 6 different genomes, have been studied using hybridization and nucleotide sequence information from the5S Dna locus. Four to five units of the major size class of 5S DNA in each species, 55 units in all, were cloned and sequenced. Both hybridization and sequence data confirmed the basic differences between the A and B, C, D genome species suggested by morphological and cytological data. The 5S DNA units of the A genome species were very similar, as were the ones from the B, C, and D genome-containing species. The 5S DNA ofO. australiensis (E genome) grouped with the B, C, D cluster, while the units ofO. brachyantha (F genome) were quite different and grouped away from all other species. 5S DNA units fromO. minuta, O. latifolia, O. australiensis, andO. brachyantha hybridized strongly, and preferentially, to the genomic DNA from which the units were isolated and hence could be useful as species/genome specific probes. The 5S DNA units fromO. sativa, O. nivara, andO. rufipogon provided A genome-specific probes as they hybridized preferentially to A genome DNA. The units fromO. punctata andO. officinalis displayed weaker preferential hybridization toO. punctata DNA, possibly reflecting their shared genome (C genome).     

Evolution of a telomere associated satellite DNA sequence in the genome ofDrosophila tristis and related species

A highly repetitive satellite DNA sequence from the genome ofDrosophila tristis with a length of 181 bp has been cloned in the pUC plasmid. The sequence hybridizes to the telomeres of all chromosomes but the Y ofD. tristis and produces a ladderlike hybridization pattern with filterbound genomic DNA ofD. tristis digested with Eco RI or Pst I with the hybridization bands at fragment lengths in multiples of 181 bp. A similar pattern is found when the genomic DNA comes fromD. ambigua or, though less clear, fromD. microlabis. Additional bands appear in the zones of high fragment lengths, too. InD. obscura andD. kitumensis, however, the 181 bp sequence is found in fragments with a length of a few kb only. The 181 bp sequence is tandemly arranged in the genome ofD. tristis and has a copy number of about 82,000 per haploid genome (i.e. 10 per cent of the total DNA). A sequence comparison among four independently cloned copies of the family fromD. tristis and another homologous sequence fromD. obscura, found by chance, shows a one to six per cent variation in basepair composition. However, low divergence (only one per cent) between two copies ofD. tristis and between the one ofD. obscura and one ofD. tristis was observed, and high divergence (six per cent) between these two pairs. This is discussed and explained as the evolutionary consequence of an existing homogenization process by unequal crossing over.     

Cloning and expression of a glucoamylase gene from Lactobacillus amylovorus ATCC 33621 in Escherichia coli

Summary The glucoamylase gene from Lactobacillus amylovorus was cloned and expressed in Escherichia coli. A genomic DNA library from Lactobacillus amylovorus was prepared by partially digesting genomic DNA with EcoRI and ligating random fragments to the EcoRI digested cloning vector, pZErO-1.1. Three E. coli transformants expressing glucoamylase were identified using a probe prepared from the STA2 glucoamylase gene from Saccharomyces cerevisiae var. diastaticus. The physical maps of the recombinant plasmids were constructed. These plasmids contained inserts of about 5.2 Kb, 5.9 Kb and 6.4 Kb respectively. Temperature and pH optima of 45°C and 6.0, respectively, were obtained for both recombinant and purified wild type glucoamylases. Also, the enzymes were found to be thermolabile at temperatures above 50°C.     

Application of LFH-PCR for the disruption ofSpoIIIE andSpoIIIG ofB. subtilis

The application of LFH-PCR (long flanking homology region-PCR) forBacillus subtilis gene disruption is presented. Without plasmid- or phage-vector construction, only by PCR, based on a DNA sequence retrieved fromB. subtilis genome data base, kanamycin resistance gene was inserted into two genes ofB. subtilis involved in sporulation,spoIIIE andspoIIIG. The effect of gene disruption on subtilisin expression was examined and the sporulation frequency of two mutants was compared to that of the host strain. For this purpose, only 2 or 3 rounds of PCR were required with 4 primers. We first demonstrated the possibility of LFH-PCR for rapid gene disruption to characterize an unknown functional gene ofB. subtilis or other prokaryote in the genomic era.     

Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

The cloning of α-amylase gene ofS. occidentalis and the construction of starch digestible strain of yeast,S. cerevisiae AS. 2. 1364 with ethanol-tolerance and without auxotrophic markers used in fermentation industry were studied. The yeast/E.coli shuttle plasmid YCEp1 partial library ofS. occidentalis DNA was constructed and α-amylase gene was screened in S.cerevisiae by amylolytic activity. Several transformants with amylolysis were obtained and one of the fusion plasmids had an about 5.0 kb inserted DNA fragment, containing the upstream and downstream sequences of α-amylase gene fromS. occidentalis. It was further confirmed by PCR and sequence determination that this 5.0 kb DNA fragment contains the whole coding sequence of α-amylase. The amylolytic test showed that when this transformant was incubated on plate of YPDS medium containing 1 % glum and 1 % starch at 30°C for 48 h starch degradation zones could be visualized by staining with iodine vapour. α-amylase activity of the culture filtratate is 740–780 mU/mL and PAGE shows that the yeast harboring fusion plasmids efficiently secreted α-amylase into the medium, and the amount of the recombinant α-amylase is more than 12% of the total proteins in the culture filtrate. These results showed that α-amylase gene can be highly expressed and efficiently secreted inS. cerevisiae AS. 2.1364, and the promotor and the terminator of α-amylase gene fromS. occidentalis work well inS. cercvisiac AS. 2.1364.     

Cloning and expression of a gene for an alpha-glucosidase from Saccharomycopsis fibuligera homologous to family GH31 of yeast glucoamylases

Cloning of cDNA encoding an α-glucosidase from the dimorphous yeast Saccharomycopsis fibuligera and characterization of the gene product were performed. The cDNA of the putative α-glucosidase gene consists of 2,886 bp, which includes an open reading frame encoding a 19 amino acid signal peptide at the N-terminal end and a 944 amino acid mature protein with a predicted molecular mass of 105.4 kDa and pI value of 4.52. The deduced amino acid sequence shows a high degree of identity (70%) with two yeast glucoamylases, namely, the extracellular glucoamylase Gam from Schwanniomyces occidentalis and the cell surface glucoamylase Gca from Candida albicans. The recombinant product, synthesized in Saccharomyces cerevisiae, is localized on the cell surface and hydrolyses maltooligosaccharides exclusively without the ability to digest soluble starch, which is consistent with the specificity characteristic of α-glucosidase, EC. 3.2.1.20.     

Use of oligonucleotide-alkaline phosphatase conjugates as non-radioactive probes for rapid analysis of a proteinase inhibitor gene fromZea mays

Conjugates of oligonucleotides and alkaline phosphatase have been prepared and used as nonradioactive hybridization probes for the study ofPis3 (=MPI) a gene encoding a proteinase inhibitor fromZea mays. Attachment of the alkaline phosphatase was carried out either at the 5′ or 3′ end of two 25-bp oligonucleotides. Sensitivity of each alkaline phosphatase-oligonucleotide probe was assessed using a chemiluminescent substrate for detection of alkaline phosphatase activity. This sensitive method allows the rapid analysis of genomic clones isolated from aZea mays library and the subsequent characterization of the completePis3 gene without the need for construction of restriction maps for the cloned DNA fragments. This general strategy may be valuable for the identification of any gene for which a limited sequence is known and for location of specific DNA sequences that represent a small region within a larger DNA fragment.     

Deoxyribonucleic acid homologies among staphylococci: Coagulase-positive reference strains

DNA hybridization results confirm the proposed separation of coagulase-positive staphylococci into two distinct species. Strains ofStaphylococcus aureus representing the various biotypes and different phage typing groups of the human biotype gave high values of reassociation with DNA fromS. aureus reference strain RN 450, at both optimal and restrictive reassociation temperatures. Similar results were obtained between strains ofS. intermedius and its reference strain K 3. Interspecific reassociation between the two coagulase-positive species was low, and each reference strain showed low DNA sequence homology with 10 coagulase-negative species.S. staphylolyticus, strain PS 73, and putative pleiotropic mutants ofS. aureus were shown to be unrelated toS. aureus.     

Molecular cloning ofTrichophyton mentagrophytes DNA sequences with promoter activity inEscherichia coli

A promoter probe library from the dermatophyte fungusTrichophyton mentagrophytes has been constructed in the pVB32 plasmid vector. Using this library, a set ofT. mentagrophytes DNA sequences with promoter activity inEscherichia coli has been cloned. The size and the resistance phenotype conferred by these DNA fragments varied. Southern blot analysis confirmed that they were derived fromT. mentagrophytes genomic DNA.     

Characterization of the amylolytic system ofCandida strains

Some physical and chemical properties ofα-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) produced in semisolid fermentation byCandida fennica FTPT-1829,C. famata FTPT-1539 andC. fennica FTPT-8903 were determined. The optimum temperature values were 42, 44, 48 °C and 60, 50, 50 °C forα-amylase and glucoamylase excreted byC. fennica 8903,C. fennica 1829 andC. famata 1539, respectively. The optimum pH values for all strains were 5.0 and 6.0 forα-amylase and glucoamylase, respectively. The degradation of pullulan by all the yeast species indicates debranching activity. This research was carried out with financial support fromPrPq/UFMG. The second author received grants fromRHAE/CNPq.     

Purification, characterization, and cloning of trimethylamine dehydrogenase fromMethylophaga sp. strain SK1

Trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde, was purified fromMethylophaga sp. strain SK1. The active TMADH was purified 12.3-fold through three purification steps. The optimal pH and temperature for enzyme activity was determined to be 8.5 and 55°C, respectively. TheV _max andK _m values were 7.9 nmol/min/mg protein and 1.5 mM. A genomic DNA of 2,983 bp fromMethylophaga sp. strain SK1 was cloned, and DNA sequencing revealed the open reading frame (ORF) of the gene coding for TMADH. The ORF contained 728 amino acids with extensive identity (82%) to that ofMethylophilus methylotrophus W₃A₁.     

Analysis of chromatin, nuclear DNA and organelle composition in somatic hybrids between Solanum tuberosum and Solanum sanctae-rosae

A protoplast fusion strategy has been applied to advance aspects of a potato breeding programme. A sub-population of somatic hybrids, selected for agronomic potential, between tetraploid Solanum tuberosum cv. Brodick and a diploid EBN2 accession, S. sanctae-rosae was subjected to detailed molecular analysis. This study reports the use of simple sequence repeats (SSRs) to identify nuclear hybrid genomes and PCR and DNA-DNA analysis to determine organelle composition in somatic hybrids derived from these parents. SSR analysis revealed somatic hybrids containing the genetic background of S. tuberosum cv. Brodick with some specific markers from S. sanctae-rosae. One somatic hybrid contained the chloroplasts derived from S. sanctae-rosae, and several hybrids had detectable RFLP mitochrondrial DNA profiles, indicating genetic re-arrangements. We also examined the use of DNase I sensitivity to the genomic and ribosomal RNA sequences in these somatic hybrids as an indicator of changes in chromatin structure. Chromatin and DNAse I analysis showed differential sensitivity to increasing levels of nuclease; DNA from several somatic hybrids was found to be resistant to DNase I compared to the parental plants. The significance of the findings to somatic cell genetics and plant breeding studies is discussed. Received: 6 July 1999 / Accepted: 29 February 2000     

Cloning and expression inEscherichia coli of arecA-like gene fromZymomonas mobilis

Intergeneric complementation ofEscherichia coli recA mutants was used to identify recombinant plasmids, within a genomic library derived fromZymomonas mobilis, that carryZ. mobilis recA-like gene. Screening of 1100 individualE. coli strains revealed four clones expressing therecA⁺ character. On restriction analysis, all four recombinant plasmids were found to be related and to exhibit a common 6.7-kb fragment. Consequently, one of the four recombinant plasmids, pZR27, was selected for further characterization. When introduced intoE. coli recA mutants, pZR27 restored resistance to methyl methane sulfonate, mitomycin-C, and UV irradiation, as well as recombination proficiency when measured by standard Hfr-mediated conjugation. The clonedrecA-like gene also restored the spontaneous and mitomycin-C-induced phage production. The origin of the insert in pZR27 from the chromosome ofZ. mobilis was confirmed by Southern transfer and DNA hybridization. However, no homology was found between therecA ofE. coli andZ. mobilis chromosomal insert DNA. TheZ. mobilis recA-like gene also encoded a major polypeptide of 38-kDa on SDS-PAGE.     

The gene (lacA) encoding galactoside acetyltransferase fromLactococcus lactis

Summary This paper reports the cloning and characterization of a gene encoding galactoside acetyltransferase from a strain ofLactococcus lactis. AP stI library ofL. lactis strain ATCC7962 DNA was constructed in plasmid pUC18. A clone harbouring a 10 kbp DNA fragment containing part of thelac operon was isolated using a labelled probe generated by PCR. DNA sequence analysis revealed the presence of a gene encoding a protein with 64.5% similarity to the galactoside acetyltransferase fromEscherichia coli. The codon usage pattern of this gene was not typical of lactococcal genes. The lactococcallac operon organization appears to be different to that of other organisms.