Pro-crossover factors regulate damage-dependent apoptosis in the Caenorhabditis elegans germ line.

During meiosis, DNA double-strand breaks (DSBs) are physiologically induced to start the recombination process and promote the formation of interhomologue crossovers (COs), which are required to ensure faithful chromosome segregation into the gametes. The timely repair of DSBs is an essential part of the meiotic programme, as accumulation of unprocessed DSBs during the pachytene stage of meiotic prophase triggers a DNA damage checkpoint response that induces apoptosis of damaged cells. We show that CO-promoting factors MSH-4, MSH-5, and ZHP-3, but not COSA-1, are required for the apoptotic response of the meiotic DNA damage checkpoint. Lack of MSH-4 or MSH-5 suppresses the apoptotic response observed in some DNA repair-defective mutants such as fcd-2 and brc-1 (orthologues of FANCD2 and BRCA1), irrespectively of the amount of DSBs present in pachytene nuclei. Although ionizing radiation fails to induce apoptosis in msh-4/5-mutant backgrounds, it induces transcriptional activation of the apoptosis-activator egl-1, which is controlled by the Caenorhabditis elegans p53 orthologue CEP-1. This finding suggests that MSH-4/5 involvement in the apoptotic response occurs downstream or independently of damage sensing and checkpoint activation. This study establishes a role for pro-CO factors MSH-4/5 and ZHP-3 in the execution of apoptosis at late meiotic prophase following the accumulation of exogenous or endogenous DNA damage.

The Caenorhabditis elegans RAD51 homolog is transcribed into two alternative mRNAs potentially encoding proteins of different sizes.

In prokaryotes, the RecA protein plays a pivotal role in homologous recombination, catalyzing the transfer of a single DNA strand into an homologous molecule. Structural homologs of the bacterial RecA protein, called Rad51, have been found in different eukaryotes (from yeast to man), suggesting a certain level of conservation in recombination pathways among living organisms. We have cloned the homolog of RAD51 in Caenorhabditis elegans. The CeRAD51 gene is transcribed into two alternative mRNAs and potentially codes for two proteins of 395 and 357 amino acids in length, respectively. We discuss the evolutionary implications of these findings.

A repetitive DNA family, conserved throughout the evolution of free-living nematodes.

This paper concerns the molecular evolution of a tandemly repeated DNA family, RcC9, originally identified in Caenorhabditis elegans. The minimum unit of periodicity of this family is the pentanucleotide [nGAAn] and its complement [nTTCn] recurring several times in alternating tandem arrays. This consensus sequence is identical to that of the heat-shock element (HSE). Multiple HSEs are present in the regulatory regions of heat-inducible genes in a wide range of eukaryotic species; HSEs mediate transcriptional activation through the binding of the heat-shock factor (HSF). I describe some repeated DNA families sharing this same consensus and found in nematode species other than C. elegans. Although the consensus is conserved, the repeated sequence diverged between species to the point that cross-hybridization is abolished. Evolutionary implications will be discussed.

Repetitive DNA sequences located in the terminal portion of the Caenorhabditis elegans chromosomes.

We describe the distribution along the chromosomes of Caenorhabditis elegans of two repetitive DNA families, RcS5 and Cerep3 and interstitial telomeric sequences. Both families show, among other interesting features, a preferential location in the terminal 30% of the chromosomes. It is known that in these regions of the genome the frequency of recombination is much higher than in the central portion, genes are rarer and sequences important for chromosome disjunction may lie.

Molecular and genomic organization of clusters of repetitive DNA sequences in Caenorhabditis elegans.

Repetitive sequences in Caenorhabditis elegans are interspersed along the holocentric chromosomes. We have physically mapped some of these repetitive families and found that, although the distribution of members of each family is relatively even along the chromosomes, members of more than one family tend to cluster in some locations. We compared the sequence organization of 11 clusters located at known positions on different chromosomes in the N2 strain. These studies allow a comparison between repetitive elements belonging to the same family that are located on the same or on different chromosomes, providing an important tool in the study of genome turnover and evolution.

Expression of the thermostable beta-galactosidase gene from the archaebacterium Sulfolobus solfataricus in Saccharomyces cerevisiae and characterization of a new inducible promoter for heterologous expression.

The lacS gene from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus encodes an enzyme with beta-galactosidase activity that, like other enzymes from this organism, is exceptionally thermophilic (optimal activity above 90 degrees C), thermostable, and resistant to common protein denaturants and proteases. Expression of the gene in mesophilic hosts is needed to uncover the molecular nature of these features. We have obtained expression of beta-galactosidase in Saccharomyces cerevisiae under the control of the galactose-inducible upstream activating sequence of the yeast genes GAL1 and GAL10. The expressed enzyme is identical in molecular mass, thermostability, and thermophilicity to the native enzyme, showing that these features are intrinsic to the primary structure of the enzyme. We also present a new promoter for the expression of thermostable proteins in S. cerevisiae. This promoter contains a sequence isolated from the nematode Caenorhabditis elegans that works as a strong, heat-inducible upstream activating sequence in S. cerevisiae. Transcription of the lacS gene under the control of this sequence is rapidly and efficiently induced by heat shock. The availability of a plate assay for monitoring beta-galactosidase activity in S. cerevisiae may allow screening for mutants affecting the efficiency and activity of the enzyme.

Use of repetitive DNA probes as physical mapping strategy in Caenorhabditis elegans.

A method for linking genomic sequences cloned in yeast artificial chromosomes (YACs) has been tested using Caenorhabditis elegans as a model system. Yeast clones carrying YACs with repeated sequences were selected from a C. elegans genomic library, total DNA was digested with restriction enzymes, transferred to nylon membranes and probed with a variety of repetitive DNA probes. YAC clones that overlap share common bands with one or more repetitive DNA probes. In 159 YAC clones tested with one restriction enzyme and six probes 28 overlapping clones were detected. The advantages and limitations of this method for construction of YAC physical maps is discussed.

Structure, evolution and properties of a novel repetitive DNA family in Caenorhabditis elegans.

We have identified a moderately repeated DNA sequence in Caenorhabditis elegans present at least at twenty different locations in the genome. Elements of this intermingled repetitive DNA family are made up of tandem subreapeats whose smaller unit is ten base pairs long. The occurrence of single base changes between units is reminiscent of mammalian satellite DNA. Sequence analysis has shown that the consensus of these repeats is identical to the consensus of the heat-shock element (HSE) common to all eukaryotes (C--GAA--TTC--G). This consensus in our sequences is repeated in tandem with an overlap of four bases (C--GAA--TTC--GAA--TTC...). We studied in detail one cloned element of the family and we were unable to detect transcription in the flanking regions either under normal growth or after heat induction. Nevertheless a 242 bp sequences out of this same element was sufficient, when located on a multicopy plasmid in Saccharomyces cerevisiae, to drive transcription from a downstream gene under heat shock conditions.

Molecular analysis of the heterogeneity region of the human ribosomal spacer.

The human ribosomal non-transcribed spacers are 30 X 10(3) base-pairs (or 30 kb) in length with a limited length heterogeneity localized in a specific region downstream from the 3' end of the transcribed region. Total DNA digested with EcoRI and BamHI and hybridized with a probe containing the 3' end of the 28 S ribosomal RNA coding region shows four major bands of 3.9 kb, 4.6 kb, 5.4 kb and 6.2 kb. The 5.4 kb band is the most abundant in every individual, followed by the 4.6 kb band. The longest and the shortest size classes are less well-represented and may even be absent. Every individual shows his own pattern of relative abundance of non-transcribed spacer length classes that can be followed through generations. We decided to investigate the molecular structure of the heterogeneity region, in order to cast light onto the mechanisms underlying the origin and maintenance of this length heterogeneity. Pertinent spacer regions of eight ribosomal clones from two human genomic libraries were subcloned and analyzed by restriction mapping and nucleotide sequencing. In the minimal length class, there is a sequence of 700 base-pairs that appears to be tandemly duplicated once, twice or three times in the other length classes. This repeated DNA module contains a region consisting of repetitions of simple pyrimidine groups like C-T, C-T-T-T or C-C-C-T. DNA module repeats may differ by the length of this pyrimidine-rich region. However, these length variations are not continuous, as revealed by Southern transfer analysis of several individuals and different cloned gene units: instead, the repeated modules fall into two discrete length classes of about 700 base-pairs and 800 base-pairs. An imperfect duplication of a short sequence of 86/89 base-pairs is present at the boundary between the heterogeneity region and the upstream flanking region, representing a very ancient duplication event.

Nucleotide sequence of a complete ribosomal spacer of D. melanogaster.

We determined the nucleotide sequence of a D. melanogaster ribosomal DNA spacer. Sequences of various portions of different cloned ribosomal spacers have been previously reported. We extend the analysis to cover the entire nontranscribed and external transcribed regions. Comparison to other cloned ribosomal DNA gene units of this species confirms a conserved general organization of the ribosomal spacer through different size classes. D. melanogaster ribosomal gene units interrupted by insertions are known to be transcribed at a much lower level than the continuous gene units. Nonetheless previous sequence analysis of a region around the transcription initiation site did not reveal significant differences in rDNA genes with and without insertions. We extend such analysis to cover the last two promoter duplications in the spacer and the entire external transcribed spacer up to the 5' cleavage site of the 18S rRNA.

Regulation of rRNA gene number in Drosophila melanogaster: new aspects resulting from the use of free duplications.

We have isolated a bobbed (bb) mutant on the free duplication Dp(1;f)122bb(+) and we have measured the rDNA content of the bb(+) and the bb loci in genetic combinations in which none of the phenomena involved in the change of the rDNA redundancy occurs. We have also measured the rDNA content of the two bb loci carried by the free duplications in two different genetic combinations: (1) XXNO(-)/Dp122bb(+) and XXNO(-)/Dp122bb females in which there are two attached X chromosomes completely deleted for the nucleolus organizer (NO) regions and therefore the only rDNA is contributed by the free duplication; (2) X/Dp122bb(+) and X/Dp122bb males, in which there are two bb loci, one on the X chromosome and the other on the X free duplication. The bb(+) and the bb duplications produced an overall increase of the rDNA content in the two genetic conditions tested. These results are not in favour of both a cis and trans effect of the regulator locus (cr(+) locus) hypothesised as being involved in the disproportionate replication of rRNA genes.

An agarose gel resolving a wide range of DNA fragment lengths.

To resolve DNA fragments ranging from several kilobases to some tens of base pairs in length, an agarose slab gel of steadily increasing thickness has been designed. During electrophoresis a gradient of decreasing electric-field strength is generated throughout the gel from the cathode end to the anode end. Shorter fragments which migrate further are decelerated, resulting in an increased linearity of the relationship between mobility and molecular weight.

DNaseI-hypersensitive sites at promoter-like sequences in the spacer of Xenopus laevis and Xenopus borealis ribosomal DNA.

We have detected a DNAseI hypersensitive site in the ribosomal DNA spacer of Xenopus laevis and Xenopus borealis. The site is present in blood and embryonic nuclei of each species. In interspecies hybrids, however, the site is absent in unexpressed borealis rDNA, but is present normally in expressed laevis rDNA. Hypersensitive sites are located well upstream (over lkb) of the pre-ribosomal RNA promoter. Sequencing of the hypersensitive region in borealis rDNA, however, shows extensive homology with the promoter sequence, and with the hypersensitive region in X. laevis. Of two promoter-like duplications in each spacer, only the most upstream copy is associated with hypersensitivity to DNAaseI. Unlike DNAaseI, Endo R. MspI digests the rDNA of laevis blood nuclei at a domain extending downstream from the hypersensitive site to near the 40S promoter. Since the organisation of conserved sequence elements within this "proximal domain" is similar in three Xenopus species whose spacers have otherwise evolved rapidly, we conclude that this domain plays an important role in rDNA function.

Comparison between rDNA magnification and bb lethal mutation frequencies in Drosophila melanogaster.

Unequal mitotic sister strand crossing over has been evoked to explain the occurrence of phenotypically bb+ males in the progeny of phenotypically bobbed males during magnification. If this is the case, complementary bbl loci should be obtained together with the bb+. To test this hypothesis we compared the frequency of bb lethal mutations in the sperms of bb males with the percentages of phenotypically bb+ males obtained during magnification of these bb males. We then compared these values with those occurring in phenotypically bb+ control males. We found that, while the number of bb+ males obtained during magnification, though variable, is high, the bb lethal mutation occurs at a very low frequency in all the genetic conditions, whatever the phenotype of the parental male.