Delta-9 fatty acid desaturase gene containing a carboxyl-terminal cytochrome b5 domain from the red alga Cyanidioschyzon merolae

A delta-9 fatty acid desaturase gene, homologous to animal and fungal acyl-coenzyme A (CoA) desaturases, was isolated from the red alga Cyanidioschyzon merolae using a degenerate PCR strategy. This gene, designated as CmFAD9, has no intron. The encoded delta-9 fatty acid desaturase (CmFad9p) consists of 476 amino acids and has an estimated molecular mass of 55.4 kDa. CmFad9p is a unique delta-9 fatty acid desaturase among plants, in that it is fused with the cytochrome b5 domain at its carboxyl terminus. This is characteristic of yeast acyl-CoA desaturase. Genomic Southern hybridization suggested that the C. merolae genome contains a single gene for delta-9 fatty acid desaturase of the animal and fungal type. Southern hybridization combined with pulsed-field gel electrophoresis revealed that CmFAD9 is probably located on chromosome XI of the 17 C. merolae chromosomes. A 1.6-kb product of this gene was transcribed throughout a light/dark synchronization culture. The discovery of CmFAD9 indicates the existence of a novel type of plant delta-9 fatty acid desaturase that may function in the endoplasmic reticulum, but not in the plastid. Received: 6 October / 22 December 1997     

Electrophoretic analysis of the nuclear and organellar genomes in the ultra-small alga Cyanidioschyzon merolae

Electrophoretic analysis reveals that the nucleus of the ultra-small eukaryotic alga Cyanidioschyzon merolae contains approximately 11.7×10⁶ base pairs (11.7 Mb) of DNA. This compact genome is fragmented into 15 small chromosomes ranging in size from 410 to 1700 kb. The migratory behaviour of chloroplast DNA is consistent with the presence of a circular plastid genome of about 170 kb. The conformation of mitochondrial DNA resembles that in yeasts and fungi and is predominantly linear and heterogenous in size.     

Electrophoretic karyotypes and gene mapping in eight species of the Fusarium sections Arthrosporiella and Sporotrichiella

Pulsed-field gel electrophoresis was used to identify karyotypes for eight species of the Fusarium sections Arthrosporiella and Sporotrichiella. The total number of chromosome-sized DNA molecules varied from six to nine, depending on the species. The sizes of chromosomes ranged from 0.4 to approximately 6.5 Mb which gave estimates of genome size of between 27.0 and 29.9 Mb. When fractionated chromosomes of the eight species were probed with Tox5, a gene coding for the key-enzyme of trichothecene biosynthesis, strong hybridization signals developed in F. poae and F. sporotrichioides, suggesting that of the eight species examined only these two have the genetic potentiality to produce trichothecene mycotoxins. By using heterologous probes from Aspergillus different rRNA loci have also been mapped on Fusarium chromosomes.     

Pulsed-field gel electrophoretic analysis of Schizophyllum commune chromosomal DNA

Summary Six chromosomal DNA bands of Schizophyllum commune have been resolved using transverse alternating field electrophoresis. The estimated sizes of the chromosomal DNAs ranged from 5.1 to 1.2 megabase pairs (Mb), the total genome size being approximately 35–36 Mb. Chromosomal length polymorphisms were found between the two S. commune isolates examined. The DNA bands corresponding to the two chromosomes containing the A and B mating-type loci were identified in hybridization experiments using probes specific to their respective linkage groups. The utility of eluted chromosomal DNAs as hybridization probes to select clones from genomic libraries, and the use of these clones in transformation experiments to identify genes of interest, are discussed.     

Genotypic differences in CC224, CC363, CC449 and CC446 of Moraxella catarrhalis isolates based on whole genome SNP,MLST and PFGE typing

Understanding the evolutionary path of M. catarrhalis from macrolide-susceptible to macrolide-resistant organism, is important for hindering macrolide resistance from propagation. Multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole genome SNP typing (WGST), as useful and practical typing tools, have both advantages and disadvantages.We studied the utility of these 3 typing methods, including the level of agreement, consistency and drawbacks, in characterizing M. catarrhalis clones and clonal complexes.We focused on four clonal complexes [CC224, CC363, CC449 (CCN10) and CC446 (CCN08)] and found that PFGE and WGST had a high level of agreement and a proper consistency of the same clone or very closely related clones, while MLST is less discriminatory for different clones. Furthermore, we also established an evolutionary distance cut-off value for “The same clone”. Moreover, we detected macrolide-resistant M. catarrhalis in CC224, which had previously been considered as a macrolide-susceptible clonal complex. A higher number of isolates belonged to ST215 compared to ST446, implying that ST215 is more likely to be the primary founder. Our study also demonstrated that all the four clonal complexes belong to the M. catarrhalis lineage 1, which is considered to be related to increased virulence potential and serum resistance. We also observed that copB II was highly related to CC449 and LOS type B was mainly confined in CC224.In conclusion, these findings provide further insight into the evolutionary characteristics of M. catarrhalis.     

Isolation and characterization of a Neurospora glucose-repressible gene

Summary Using differential hybridization, the cDNA copy of a Neurospora gene coding for an abundant glucose-repressible mRNA (grg-l) has been isolated. The cDNA was used to clone the genomic copy, and both were sequenced. The cDNA is nearly full length and contains putative translational start and termination codons. Conceptual translation indicates that grg-1 mRNA could direct the synthesis of a 7,000 molecular weight polypeptide. The genomic clone, contained in an 1,888 by PvuII fragment, encompasses the entire cDNA as well as 838 by of 5 and 369 bp of 3 flanking sequence. Comparison of the cDNA and genomic clones revealed the presence of two short introns in potential protein-coding sequences. grg-1 message levels were found to increase within minutes following the onset of glucose deprivation and rise 50 fold during the first 90 min of derepression.     

Genome analysis of imperfect fungi: electrophoretic karyotyping and characterization of the nuclear gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Curvularia lunata

Summary The gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) has been isolated from a genomic library of the filamentous fungus Curvularia lunata. The coding region of this gene consists of 1014 nucleotides and is interrupted by four introns. The gpd gene product shows a high degree of sequence identity with the corresponding proteins of various species belonging to both taxonomically related (e.g., Aspergillus nidulans), as well as more divergent, taxa. Using contour-clamped homogeneous electric field (CHEF) gel electrophoresis eight distinct chromosomal bands have been resolved, with two bands migrating as doublets and one as a triplet. Thus, the total number of chromosomes of C. lunata appears to be 12. The size of the chromosomes ranges from about 1.4 Mb to 4.0 Mb allowing an estimation of the genome to be approximately 29.7 Mb. By hybridization of fractionated chromosomes the gpd gene and the rDNA locus have been localized on individual chromosomes.     

Isolation,characterization and mapping of pyrimidine auxotrophs of Phycomyces blakesleeanus

A total of seven pyrimidine auxotrophs of Phycomyces were isolated from among 5-fluoroorotate acid (5-FOA)-resistant mutants. They were classified by complementation into two groups. A representative mutant strain belonging to one group was deficient in orotate phosphoribosyl transferase (OPRTase; EC 2.4.2.10) activity; the mutant strain belonging to the second group was deficient in orotidine-5-monophosphate decarboxylase (OMPdecase; EC 4.1.1.23). These mutants are defective in the genes pyrF and pyrG respectively. The results from random spore analysis, tetrad analysis, and gene-centromere distances showed that these two markers are located in linkage group VI, with pyrG being a proximal marker and pyrF a distal one.     

Isolation,sequencing, and characterization of crp-5, a gene encoding a Neurospora ribosomal protein

A Neurospora crassa cytoplasmic ribosomal protein gene, crp-5, has been isolated and characterized. The cDNA was isolated by a differential screening of a cDNA library for glucose-inducible genes. The cDNA was subsequently used to identify and isolate crp-5 genomic sequences. Computer analysis of the DNA sequences showed that they contain an open reading frame which encodes a protein homologous to the rat ribosomal protein S26. The crp-5 mRNA levels are regulated in a carbon-source-dependent manner. The organization of the gene and the region upstream of the coding sequences are discussed.     

Cloning and characterization of the nuclear gene and cDNAs for triosephosphate isomerase of the marine red alga Gracilaria verrucosa

cDNAs and an intronless single-copy nuclear gene (TPI1) encoding triosephosphate isomerase have been cloned and sequenced from the marine red alga Gracilaria verrucosa. The predicted amino-acid sequence of TPI1 is readily alignable with those of other known TPIs; 26 of 27 active-site residues and 19 of 26 intersubunit-contact residues are identical between TPIs of G. verrucosa and/or animals and green plants. A partial cDNA sequence of a second TPI gene (TPI2), presumably encoding plastid-localized TPI, was recovered by PCR and demonstrated by phylogenetic analysis to be red algal; no TPI2 cDNA or genomic clones could be recovered. Genomic Southern analysis demonstrated that at least two TPI-like genes are present in the nuclear DNA of G. verrucosa.Issued as NRCC no. 38070 GSDB accession L38662     

cDNA cloning and characterization of the nuclear gene encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase from the marine red alga Gracilaria verrucosa

Using a PCR-generated homologous probe, we have recovered a cDNA (GapA cDNA) encoding the complete 338 amino-acid chloroplast GAPDH of the marine red aga Gracilaria verrucosa, together with its 78 amino-acid transit peptide. This cDNA was readily aligned with chloroplast-localized GAPDH genes (GapA) and (GapB) of green plants. The proline residue which contributes to the specificity of NAD⁺ binding to cytosolic GAPDHs is absent from the deduced polypeptide chain of G. verrucosa GapA as is also the case in the chloroplast GAPDHs of plants. The transit peptide shows a high proportion of random coil, an amino-terminal Met-Ala dipeptide, a high content of hydroxylamino acids, and a net positive charge. The polyadenylation signal appears to the AGTAAA. Genomic Southernhybridization data indicate that only one chloroplast-GAPDH gene may occur in G. verrucosa. Bootstrapped parsimony trees indicate that the G. verrucosa Gap A gene is a sister group to plant chloroplast-GAPDH genes, and are most readily interpreted as showing that red algal and plant chloroplast-localized GAPDHs arose in a single endosymbiotic event.     

Antimicrobial resistance and molecular analysis of non-typhoidal Salmonella isolates from human in Tunisia

During the period from 2006 to 2007, a total of 32 clinical isolates of Salmonella enterica were isolated from diarrheagenic stool samples and further examined for their susceptibility to various antibiotics. Sixteen of the human isolates were from the capital Tunis, 11 were from Sousse, four were from Nabeul and one was from Mahdia, Tunisia. The isolates were serotyped and identified at the National Centre of Enteropathogenic Bacteria, Pasteur Institute, Tunis (Centre National de Salmonella, Shigella et Vibrio – Institut pasteur de Tunis); nine distinct serovars were identified: Enteritidis (n = 20), Typhimurium (n = 4), Zanzibar (n = 2), Manhattan (n = 1), Bovismorbificans (n = 1), Amsterdam (n = 1), Saint Paul (n = 1), Kentucky (n = 1) and Muenster (n = 1). Our results showed that 25 Salmonella isolates (78.1 %) were resistant to antibiotics with 20 isolates (62.5 %) displayed resistance to ampicillin. Isolates sharing invA gene, as shown by PCR amplification, were further characterized by the techniques of pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI and plasmid analysis to determine possible genetic relationships among Salmonella enterica clinical isolates and to assess genetic diversity. Plasmid profiling identified seven plasmid profiles (with 1–5 plasmids) among the isolates; four isolates (Salmonella Kentucky, Salmonella Muenster, Salmonella Bovismorbificans and Salmonella Zanzibar) did not carry any plasmid. The isolates were differentiated into 10 distinct XbaI-pulsotypes. Our findings show genetic diversity among the different serovars and cluster analysis of compiled serotyping, PFGE, plasmid profiling and antibiotic resistance data provided additional discrimination.     

Construction, characterization and chromosomal mapping of bacterial artificial chromosome (BAC) library of Yunnan snub-nosed monkey (Rhinopithecus bieti

We constructed a high redundancy bacterial artificial chromosome library of a seriously endangered Old World Monkey, the Yunnan snub-nosed monkey (Rhinopithecus bieti) from China. This library contains a total of 136 320 BAC clones. The average insert size of BAC clones was estimated to be 148 kb. The percentage of small inserts (50-100 kb) is 2.74%, and only 2.67% non-recombinant clones were observed. Assuming a similar genome size with closely related primate species, the Yunnan snub-nosed monkey BAC library has at least six times the genome coverage. By end sequencing of randomly selected BAC clones, we generated 201 sequence tags for the library. A total of 139 end-sequenced BAC clones were mapped onto the chromosomes of Yunnan snub-nosed monkey by fluorescence in-situ hybridization, demonstrating a high degree of synteny conservation between humans and Yunnan snub-nosed monkeys. Blast search against human genome showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the BAC library. This library and the mapped BAC clones will serve as a valuable resource in comparative genomics studies and large-scale genome sequencing of nonhuman primates. The DNA sequence data reported in this paper were deposited in GenBank and assigned the accession number CG891489-CG891703.     

Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. marcospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5 Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora     

Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene,XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant

Summary A P. stipitis cDNA library in gt11 was screened using antisera against P. stipitis xylose reductase and xylitol dehydrogenase, respectively. The resulting cDNA clones served as probes for screening a P. stipitis genomic library. The genomic XYL2 gene was isolated and the nucleotide sequence of the 1089 bp structural gene, and of adjacent non-coding regions, was determined. The XYL2 open-reading frame codes for a protein of 363 amino acids with a predicted molecular mass of 38.5 kDa. The XYL2 gene is actively expressed in S. cerevisiae transformants. S. cerevisiae cells transformed with a plasmid, pRD1, containing both the xylose reductase gene (XYL1) and the xylitol dehydrogenase gene (XYL2), were able to grow on xylose as a sole carbon source. In contrast to aerobic glucose metabolism, S. cerevisiae XYL1-XYL2 transformants utilize xylose almost entirely oxidatively.