Caenorhabditis elegans Ce-rdh-1/rad-51 functions after double-strand break formation of meiotic recombination

During meiotic prophase 1, homologous recombination is accompanied by dynamic chromosomal changes. The Ce-rdh-1/rad-51 gene is the only bacterial recA-like gene in the nematode C. elegans genome. Upon depletion of Ce-rdh-1/rad-51 using the RNA interference method, abnormal kinked chromosomes can be observed in mature oocytes at diakinesis, whereas synapsis between homologous chromosomes during the pachytene stage is normal. Following fertilization, Ce-rdh-1/rad-51-depleted embryos die early in embryogenesis, and their nuclei exhibit abnormal chromosome fragments and bridges. From epistasis analyses with Ce-spo-11 defective mutant and ionizing radiation, it is indicated that Ce-rdh-1/rad-51 functions after double-strand break (DSB) formation of meiotic recombination. Under the Ce-chk-2 defective condition, whose meiotic synapsis and meiotic recombination between homologous chromosomes are completely inhibited, the Ce-rdh-1/rad51 is normally expressed in the gonadal cells. Moreover, it seems that exogenous DSBs in the Ce-chk-2 defective nuclei at the pachytene stage can be repaired between sister chromatids in a Ce-rdh-1/rad-51-dependent manner. These results indicate that Ce-rdh-1/rad51 functions after both endogenous and exogenous DSB formation during meiosis, but not as pairing centers for meiotic synapsis.     

Synchronized meiosis and recombination in fission yeast: observations with pat1-114 diploid cells

Summary The mutation pat1-114 has been used to synchronize meiosis in the fission yeast Schizosaccharomyces pombe. We have investigated several aspects of such synchronized meiotic cultures. In both pat1-114 and pat1 ⁺ diploids, meiotic landmark events are initiated at the same time after meiosis induction, but synchrony is much more pronounced in the pat1-114-driven meiosis. Commitment to recombination and to meiosis have been timed at 2 h after meiotic induction. Due to a seven-fold reduction of intragenic recombination frequency in the ade6 region of pat1-114 diploids, physical analysis of recombination has not been possible. We have distinguished three factors that influence intragenic recombination frequencies: temperature, azygotic versus zygotic meiosis, and the nature of the pat1 allele. Differences and similarities in the timing of meiotic landmarks in S. cerevisiae and S. pombe are discussed.     

Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an unusual structure and arrangement

Summary Nucleotide sequence analysis of a 17043 basepair (bp) region of the Chlamydomonas reinhardtii plastome indicates the presence of three open reading frames (ORFs) similar to RNA polymerase subunit genes. Two, termed rpoB1 and rpoB2, are homologous to the 5-and 3-halves of the Escherichia coli beta subunit gene, respectively. A third, termed rpoC2, is similar to the 3-half of the bacterial beta' subunit gene. These genes exhibit several unusual features: (1) all three represent chimeric structures in which RNA polymerase gene sequences are juxtaposed in-frame with long sequences of unknown identity; (2) unlike their counterparts in plants and eubacteria, rpoB1 and rpoB2 are separated from rpoC2 by a long (7 kilobase-pair, kbp) region containing genes unrelated to RNA polymerase; (3) DNA homologous to the 5 half of rpoC (termed rpoC1 in other species) is not present at the 5 end of rpoC2 and could not be detected in C. reinhardtii chloroplast DNA. RNA expression could not be detected for any of the RNA polymerase genes, suggesting that they are pseudogenes or genes expressed at stages of the C. reinhardtii life-cycle not investigated. The three genes are flanked by GC-rich repeat elements. We suggest that repeat DNA-mediated chloroplast recombination events may have contributed to their unusual arrangement.     

Isolation of early meiotic recombination genes analogous to S. cerevisiae REC104 from the yeasts S. paradoxus and S. pastorianus

The REC104 gene of Saccharomyces cerevisiae is required to initiate recombination in meiosis. Mutations in REC104 eliminate meiotic recombination and lead to the production of inviable spores. To determine if analogous genes exist in other yeasts, clones that hybridized to a REC104 probe were isolated from the yeasts S.␣paradoxus and S.␣pastorianus. When transformed into a rec104 strain, the REC104 analogs from these two yeasts restored spore viability and meiotic recombination to the same level as a REC104 gene cloned from S.␣cerevisiae. Compared to S.␣cerevisiae, the S.␣paradoxus gene codes for 79% identical amino acids and has 86% nucleic-acid identity in the promoter region and 84% in the coding region. The S.␣pastorianus gene codes for 63% identical amino acids and has 59% and 71% identity in the promoter and the coding regions, respectively. Received: 19 August / 19 September 1996     

Organization and structure of plastome psbF,psbL, petG and ORF712 genes in Chlamydomonas reinhardtii

Summary We have determined the nucleotide sequence of a 5159 base-pair (bp) region of the Chlamydomonas reinhardtii plastome containing three photoelectron transport genes, psbF, psbL and petG, and an unusual open reading frame, ORF712. The photosynthetic genes have an unprecedented arrangement. psbF and psbL are located in close proximity to petG, and are not grouped with two other genes of the cytochrome b₅₅₉ locus, psbE and ORF42. ORF712, located adjacent to psbL, has homology at its 5-and 3-ends to the ribosomal protein rps3 gene, but contains, a central 437 residue domain that lacks similarity to any other known sequence. These sequences add to the growing body of evidence that the chloroplast genome of C. reinhardtii has a significantly different gene arrangement to its counterpart in plants. The structure of ORF712 also provides another example of a phenomenon we have discovered with C. reinhardtii RNA polymerase genes (Fong and Surzycki 1992); namely, that the algal plastome contains chimeric genes in which reading frames with homology to known genes are juxtaposed in-frame with long coding regions of unknown identity.     

Putative gene promoter sequences in the chlorella viruses

Three short (7 to 9 nucleotides) highly conserved nucleotide sequences were identified in the putative promoter regions (150 bp upstream and 50 bp downstream of the ATG translation start site) of three members of the genus Chlorovirus, family Phycodnaviridae. Most of these sequences occurred in similar locations within the defined promoter regions. The sequence and location of the motifs were often conserved among homologous ORFs within the Chlorovirus family. One of these conserved sequences (AATGACA) is predominately associated with genes expressed early in virus replication.     

The Chlorella H+/hexose cotransporter gene

Summary The complete genomic sequence of the inducible Chlorella kessleri H⁺/hexose cotransporter (HUP1) has been obtained from two overlapping clones isolated from a gt10 library. The HUP1 gene is interrupted by 14 introns with the first intron being located in the 5-untranslated part of the gene. The average intron length is 220 bp, yielding a very regular intron/exon pattern in the gene. The codon usage in this gene is strongly biased with a clear preference for C and a strong suppression of A. A consensus sequence for a putative algal polyadenylation sequence is shown and compared with other algal cDNA sequences.     

A truncated recombination repeat in the mitochondrial genome of a Petunia CMS line

Repeated sequences known as recombination repeats are present in the majority of plant mitochondrial genomes. Two recombination repeat sequences from Petunia have been analyzed. The two repeats are virtually identical over 1.42 kb. One of the repeats is truncated and is likely to have arisen from a rare recombination event in the full-length repeat. Two sequence-blocks within the Petunia repeat are highly similar to sequences in the 5 flank of several plant mitochondrial genes. No sequence motifs are shared by the Petunia repeat and other sequenced plant mitochondrial recombination repeats, suggesting that the recombination occurs by an homologous, rather than a site-specific, mechanism.     

The two genes for the P700 chlorophyll a apoproteins on the Euglena gracilis chloroplast genome contain multiple introns

Summary We have determined the complete nucleotide sequence of the two genes encoding the P₇₀₀ chlorophyll a apoproteins of the photosystem I reaction center of the Euglena gracilis chloroplast genome. The two genes are separated by 77 bp, are of the same polarity, and span a region which is greater than 9.0 kbp. The psaA gene (751 codons) is interrupted by three introns and the psaB gene (734 codons) by six introns. The introns range in size from 361 to 590 bp, whereas the exons range in size from 42 to 1,194 bp. The introns are extremely AT rich with a pronounced base bias of T > A > G > C in the RNA-like strand. Like other interrupted protein genes in the Euglena chloroplast genome, the psaA and psaB introns are similar to mitochondrial group II introns in having the splice junction consensus sequence, 5 GTGCGNTTCG ..... INTRON ..... TTAATTTTAT 3 and conserved secondary structural features. Except for the placement of the first intron, the intron-exon organization of these two highly homologous genes is not conserved. The other introns fall at or near putative surface domains of the predicted gene products. The psaA and psaB gene products are 74% homologous to one another and 93% and 95% homologous, respectively, to the psaA and psaB gene products of higher plant chloroplasts. The predicted secondary structure derived from the primary amino acid sequence has 11 potential membrane-spanning domains. Abbreviations and notations: Gene names follow the convention of Hallick and Bottomley (1983: psaA, psaB, genes for the P₇₀₀ apoprotein; psbE an psbF, genes foe the subunits of cytochrome b ₅₅₉; orfN, open reading frame of N condons     

Genetic evidence for different RAD52-dependent intrachromosomal recombination pathways in Saccharomyces cerevisiae

Mutations in the RecA-like genes RAD51 and RAD57 reduce the frequency of gene conversion/reciprocal exchange between inverted repeats 7-fold. However, they enhance the frequency of deletions between direct repeats 5–12-fold. These induced deletions are RAD1- and RAD52-dependent. On the basis of these results it is proposed that there are several RAD52-dependent pathways of recombination: the recombinational repair pathway of gene conversion/reciprocal exchange dependent on RAD51 and RAD57; a RAD1-and RAD52-dependent pathway exclusively responsible for deletions that are induced in rad51 and rad57 mutants; and finally, it is possible that the gene conversion/reciprocal exchange events observed in rad51 and rad57 strains represent another RAD52-dependent recombination pathway of gene conversion/reciprocal exchange that does not require Rad51 and Rad57 functions. It is also shown that the RAD10 excision-repair gene is involved in long gene conversion tracts in homologous recombination between inverted repeats, as previously observed for RAD1. Finally, an analysis of meiotic recombination reveals that deletions are induced in meiosis 100-fold above mitotic levels, similar to intrachromosomal gene conversion/reciprocal exchange, and that, in contrast to intrachromosomal meiotic gene conversion (50% association), intrachromosomal meiotic gene conversion is not preferentially associated with reciprocal exchange (12–30% of association).     

Identification and characterization of odv-e25 of Spodoptera litura multicapsid nucleopolyhedrovirus

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) odv-e25 is 684 bp long, potentially encoding 227 amino acids with a predicted molecular weight of 24.9 kDa. Homology analysis indicated that SpltMNPV ODV-E25 has 35–65% amino acid identity with that of other known baculoviruses. RT-PCR results revealed that the odv-e25 is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (TTAAG). Western blot analysis of odv-e25 expression with an antiserum made against 6 × His tagged ODV-E25 expressed in Escherichia coli indicated that it was present as a doublet of approximately 27 kDa from 24 h through 96 h in SpltMNPV-infected Spli-221 cells. Similar results were seen on Western blots of Spodoptera exigua (Se)MNPV-infected Se301 cells. Immunofluorescence analysis showed that ODV-E25 was predominantly present in the cytoplasm of SpltMNPV-infected cells and localized to the envelopes of occlusion-derived virus.     

Molecular structure of the SWA2 gene encoding an AMY1-related α-amylase from Schwanniomyces occidentalis

A 2.1-kb DNA fragment containing the SWA2 gene determining an -amylase from Schwanniomyces occidentalis has been sequenced. It contains an open reading frame of 1521 bp which has the potential to encode a 507 amino-acid protein of Mr 55966. Its deduced aminoacid sequence shows significant similarities to the sequence of other studied -amylases. These similarities identify a consensus sequence, F(LIV)(ED)NHD, which is shared in addition by most maltases, invertases and glucoamylases.     

LEU2 directed expression of β-galactosidase activity and phleomycin resistance in Yarrowia lipolytica

Summary The nucleotide sequence of a 968 by DNA fragment spanning the promoter and the 5 upstream sequence of the LEU2 coding sequence of the yeast Yarrowia lipolytica has been determined. A LEU2:lacZ gene fusion has been constructed and expressed in transformed yeast cells, showing that as few as 232 by of the LEU2 promotor were sufficient to direct gene expression. In order to develop new markers for transformation of this yeast, the LEU2 initiation codon was destroyed by in vitro mutagenesis and replaced by a cloning site. A gene confering phleomycin resistance in E. coli was attached to the LEU2 promoter and shown to be efficiently expressed in yeast: direct selection of phleomycin resistant transformants was possible.     

The influence of the Ustilago maydis REC1 gene on plasmid-chromosome recombination

The REC1 gene of U. maydis has an important but ill-defined role in DNA recombination and repair. We have examined its role in plasmid-chromosome recombination. Plasmid DNA was linearised at various locations with respect to the cloned U. maydis PYR3 gene and introduced into cells by transformation. Chromosomal integration and repair by an homologous cross-over with plasmid containing a double-strand break or gap in the PYR3 gene was markedly reduced in the absence of the REC1 gene product. Homologous replacement of the chromosomal pyr3-1 allele by a single copy of wild-type sequences from plasmid cut outside PYR3 was not found in the absence of the REC1 product. Instead, novel transformants generated in its absence suggests that ligation plays a role in their generation.     

The Claviceps purpurea glyceraldehyde-3-phosphate dehydrogenase gene: cloning,characterization, and use for the improvement of a dominant selection system

The GPD 1 gene of Claviceps purpurea coding for glyceraldehyde-3-phosphate dehydrogenase was cloned and sequenced, including 1,800 bp of its 5 upstream region. This gene shows an identical structure to the gpd gene of Podospora anserina and Cryphonectria parasitica (one intron at an identical position) with high homology at both the DNA and amino-acid levels. Two fragments of the promoter spanning from the ATG to-500 bp and to-1,400 bp were fused to the phleomycin-resistance gene. Both constructs transformed C. purpurea at a high rate. The enhanced expression of the long vector construct indicates the presence of additional elements between-500 bp and-1,400 bp upstream of the initiation codon.     

Characterization of genes which influence allelic recombination in Coprinus cinereus

Summary Previous work has shown that recombination between alleles of the ftr cistron is disturbed by two marker effects, one reduces the recombination frequency and the other increases it. These effects have been studied further. The results show that both enhancement and reduction are controlled by single genes which seem to be independent of one another. The genes are symbolized recE (for recombination enhancement) and recR (recombination reduction). Both genes are fully dominant, non-additive, and segregate readily from one another and from the ftr cistron. Recombination between any pair of ftr alleles is increased when recE is part of the genotype, but recE has no effect on recombination between alleles of the me-5 locus. On the other hand, the reduction of recombination caused by recR in the ftr cistron is polarised and alley-specific, but recR increases the frequency of recombination between me-5 alleles. The data are interpreted on the basis that the rec-gene products may be involved in chromatid pairing and that polymorphic variants of them cause differences in pairing which, by altering the opportunities for recombination, are observed as differences in allelic recombination frequency.     

Multiple-copy integration of the α-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis

Summary We have developed a vector system for high-copy-number integration into the ribosomal DNA of the yeast Kluyveromyces lactis. This system is analogous to the pMIRY-system developed for Saccharomyces cerevisiae. Plasmids containing a portion of K. lactis rDNA for targeted homologous recombination, as well as the S. cerevisiae TRP1 gene with various promoter deletions, were constructed and, after transformation to K. lactis, analyzed for both copy number and stability. These plasmids were found to be present in about 60 copies per cell and were stably maintained during growth under non-selective conditions. Using this vector system, we expressed a fusion construct containing the S. cerevisiae GAL7 promoter, the SUC2 (invertase) signal sequence and the gene coding for -galactosidase from the plant Cyamopsis tetragonoloba. Although the maximum copy number of these integrated plasmids was only about 15, we nevertheless obtained a high level of -galactosidase production (250 mg/l) with a secretion efficiency of about 95%. When compared to extrachromosomal K. lactis vectors containing the same fusion construct, the multicopy integrants showed a much higher -galactosidase production level and a considerably higher stability under non-selective conditions.     

A nucleotide sequence involved in replicative transformation of a filamentous fungus

Replicative plasmids generated through in-vivo recombination have been identified among transformants of the fungus Pleurotus ostreatus. In addition to sequences from a standard selection vector (pAN7-1), these recombinant plasmids contain recombined sequences of chromosomal origin conferring replicative potential upon the vector. One such recombined sequence, an 1148-bp insert into plasmid pP01, has been characterized. This sequence has been analyzed for secondary structural features as well as for consensus sites affiliated with origins of replication (ori) in other eukaryotic systems. The 1148-bp insert lacks an ORF and does not contain an acceptable match to the commonly identified 11-bp ars consensus sequence (A/TTTTATA/GTTTA/T) for autonomous replication in the yeast Saccharomyces cerevisiae. The analysis, however, revealed a cluster of three hairpin-loop-forming subsequences with individual G_25°C free energy values of-7.6,-6.4 and-5.2 kcal mol^-1. Also found were two 7-bp analogues to centromere-affiliated sequences recognized in other fungi, as well as several putative gyrase recognition sites comparable to the 9-bp S. cerevisiae/E. coli gyrase-binding consensus sequence. Sequences comparable to the ori of the yeast 2-m plasmid or to various sequences associated with ori of yeast/fungal mitochondrial DNAs (mtDNA) were not present in the 1148-bp insert. Replication of pP01 appears rather to involve a replication of chromosomal derivation devoid of an ars-type consensus.     

Influence of codon usage and translation initiation codon context in theAcNPV-based expression system: Computer analysis using homologous and heterologous genes

Codon usage by all the known gene sequences fromAutographa californica nuclear polyhedrosis virus (AcNPV) was compared with that of firefly luciferase (luc) and the beta subunit of human chorionic gonadotropin (hCG) expressed to contrasting levels in the baculovirus system. The highly expressedluc gene showed a codon usage similar toAcNPV genes, as reflected by a very low D-squared statistic value (0.78) and a similar G/C usage (45%) at wobble positions. However, the underexpressed hCG gene displayed a high D-squared value (7.3) and G/C usage (82.5%) at the wobble base positions. Alignment of the 20 nucleotides around the initiation codon of 23AcNPV genes identified a novel consensus translation initiation sequence aag/ta/tat/aa/cAAaATGaa/ct/ag/aAan, which was quite different from the Kozak consensus sequence (GCC)GCCA/GCCATGG. An extension of these analyses to a sample of other heterologous genes overexpressed and underexpressed in BEVS suggested similar trends. These theoretical analyses have important implications for heterologous gene expression in this system.     

Organization of ribosomal protein genes rp123, rp12, rps19, rp122 and rps3 on the Euglena gracilis chloroplast genome

Summary The nucleotide sequence (4,814 bp) was determined for a cluster of five ribosomal protein genes and their DNA flanking regions from the chloroplast genome of Euglena gracilis. The genes are organized as rp123 — 150 by spacer — rpl2 — 59 by spacer —rps19 — 110 by spacer — rp122 — 630 by spacer — rps3. The genes are all of the same polarity and reside 148 bp downstream from an operon for two genes of photosystem I and four genes of photosystem II. The Euglena ribosomal protein gene cluster resembles the S-10 ribosomal protein operon of Escherichia coli in gene organization and follows the exact linear order of the analogous genes in the tobacco and liverwort chloroplast genomes. The number and positions of introns in the Euglena ribosomal protein loci are different from their higher plant counterparts. The Euglena rp123, rps19 and rps3 loci are unique in that they contain three, two and two introns, respectively, whereas rp12 and rp122 lack introns. The introns found in rpl23 (106, 99 and 103 bp), rps19 (103 and 97 bp) and rps3 intron 2 (102 bp) appear to represent either a new class of chloroplast intron found only in constitutively expressed genes, or possibly a degenerate version of Euglena chloroplast group II introns. They are deficient in bases C and G and extremely rich in base T, with a base composition of 53–76% T, 25–34% A, 3–10% G and 2–7% C in the mRNA-like strand. These six introns show minimal resemblance to group IT chloroplast introns. They have a degenerate version of the group II intron conserved boundary sequences at their 5 and 3 ends. No conserved internal secondary structures are apparent. By contrast, rps3 intron 1 (409 bp) has a potential group II core secondary structure. The five genes, rpl23 (101 codons), rpl2 (278 codons), rpsl9 (95 codons), rpl22 (114 codons) and rps3 (220 codons) encode lysine-rich polypeptides with predicted molecular weights of 12,152, 31,029, 10,880, 12,819, and 25,238, respectively. The Euglena gene products are 18–50%, and 29–58% identical in primary structure to their E. coli and higher plant counterparts, respectively. Oligonucleotide sequences corresponding to Euglena chloroplast ribosome binding sites are not apparent in the intergenic regions. Inverted repeat sequences are found in the upstream flanking region of rp123 and downstream from rps3. Abreviations: Gene names follow the convention of Hallick and Bottomley (1983): rp123, rpl2, rpl22 are, respectively, genes for the L23, L2, L22 polypeptides of the 50S ribosomal subunit; rps19 and rps3 are genes for the S19 and S3 polypeptides of the 30S ribosomal subunit