CLIP-50 immunolocalization during mouse spermiogenesis suggests a role in shaping the sperm nucleus.

The spermatid nucleus and cytoplasm undergo dramatic morphological modifications during spermatid differentiation into mature sperm. Some of the external force causing this nuclear shaping is generated by a microtubular structure termed the manchette, which attaches to the perinuclear ring of the spermatid. Here, we report the isolation and characterization of a protein component of this perinuclear ring in an immunological screening of a mouse testis cDNA library. We termed this protein CLIP-50 because of its high similarity at the amino acid level to the C-terminal region of the microtubule-binding protein CLIP-170/restin. CLIP-50 lacks the characteristic microtubule-binding motif, but retains a portion of the predicted coiled-coiled domain and the metal-binding motif. The CLIP-50 transcript and protein are abundant in testis. The protein is also expressed in heart, lung, kidney, and skin. A distinct size variant exists in brain. In the spermatids, CLIP-50 protein localizes specifically to the centriolar region where the sperm tail originates and to the perinuclear ring from which the manchette emerges. CLIP-50 staining is retained in the ring throughout its migration over the surface of the nucleus which accompanies the nuclear shaping into its characteristic sperm configuration. This localization pattern indicates a very specific function for this novel CLIP derivative during mouse spermiogenesis.

Paramecium genome survey: a pilot project.

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

The genomic structure of SYCP3, a meiosis-specific gene encoding a protein of the chromosome core.

SYCP3 localizes to the lateral elements of the synaptonemal complex and is essential for male meiosis. The genomic structure of SYCP3 consists of nine exons spanning approximately 14 kb. In mouse and rat, but not in hamster, the putative translation start of SYCP3 is present in the first exon. The putative promoter of SYCP3 was also cloned and shown to drive transcription of a reporter gene in somatic cells.

Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila.

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs). Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein. We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries. A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47-81 bp from the right side of mse2.9. The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs. In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing. Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena.

Glycine metabolism in Candida albicans: characterization of the serine hydroxymethyltransferase (SHM1, SHM2) and threonine aldolase (GLY1) genes.

Genes encoding the mitochondrial (SHM1) and cytosolic (SHM2) serine hydroxymethyltransferases, and the L-threonine aldolase gene (GLY1) from Candida albicans were cloned and sequenced. All three genes are involved in glycine metabolism. The C. albicans Shm1 protein is 82% identical to that from Saccharomyces cerevisiae and 56% identical to that from Homo sapiens. The corresponding identities for the Shm2 proteins are 68% and 53%. The Gly1 protein shares significant identity with the S. cerevisiae L-threonine aldolase (55%) and also with threonine aldolases from Aeromonas jandiae (36%) and Escherichia coli (36%). Genetic ablation experiments show that GLY1 is a non-essential gene in C. albicans and that L-threonine aldolase plays a lesser role in glycine metabolism than it does in S. cerevisiae. GenBank Accession Nos of the C. albicans SHM1 and SHM2 are AF009965 and AF009966, respectively. Accession No. for C. albicans GLY1 is AF009967.

RAD51 and DMC1 form mixed complexes associated with mouse meiotic chromosome cores and synaptonemal complexes.

The eukaryotic RecA homologues RAD51 and DMC1 function in homology recognition and formation of joint-molecule recombination intermediates during yeast meiosis. The precise immunolocalization of these two proteins on the meiotic chromosomes of plants and animals has been complicated by their high degree of identity at the amino acid level. With antibodies that have been immunodepleted of cross-reactive epitopes, we demonstrate that RAD51 and DMC1 have identical distribution patterns in extracts of mouse spermatocytes in successive prophase I stages, suggesting coordinate functionality. Immunofluorescence and immunoelectron microscopy with these antibodies demonstrate colocalization of the two proteins on the meiotic chromosome cores at early prophase I. We also show that mouse RAD51 and DMC1 establish protein-protein interactions with each other and with the chromosome core component COR1(SCP3) in a two-hybrid system and in vitro binding analyses. These results suggest that the formation of a multiprotein recombination complex associated with the meiotic chromosome cores is essential for the development and fulfillment of the meiotic recombination process.

Meiotic activation of rat pachytene spermatocytes with okadaic acid: the behaviour of synaptonemal complex components SYN1/SCP1 and COR1/SCP3.

The phosphatase inhibitor okadaic acid accelerates meiotic events in rodent germ cells in culture. Isolated pachytene spermatocytes treated with okadaic acid proceed to a metaphase I arrest in a few hours as opposed to the similar process in vivo, which requires several days. Leptotene/zygotene spermatocytes cannot be activated in this way, suggesting that okadaic acid enables cells to bypass a sensor of the meiotic progression, which is pachytene specific. We monitored the chromosome behaviour accompanying the transition to metaphase I in rat spermatocytes with antibodies against COR1/SCP3, a component of the meiotic chromosome cores, and against the synaptic protein, SYN1/SCP1. Okadaic acid induced a rapid synaptonemal complex dissolution and bivalent separation, followed by chromosome condensation and chiasmata formation, similar to the succession of events in untreated cells. The similarity between meiosis I induced with okadaic acid and the meiosis I events in vivo extends to the dissolution of the nuclear membrane and the disappearance of the microtubule network at the onset of metaphase I. This cell culture system provides a model for the in vivo transition from pachytene to metaphase I and therefore can be used in the study of this transition at the molecular level. The effect of okadaic acid is most likely mediated by the activation of tyrosine kinases, as addition of genistein, a general tyrosine kinase inhibitor, completely abolishes the observed effect of okadaic acid on chromosome metabolism. The okadaic acid-induced progression to the metaphase I arrest is not affected by the inhibition of protein synthesis. However, pachytene spermatocytes incubated in the presence of protein synthesis inhibitors for 6 hours show loss of synapsis which is abnormal in that it is not accompanied by chiasmata formation. The two meiosis-specific proteins, SYN1/SCP1 and COR1/SCP3, are efficiently phosphorylated in vitro by extracts from isolated pachytene cells. Extracts from cells that have reached metaphase I upon okadaic acid treatment, with concomitant displacement of SYN1/SCP1 and COR1/SCP3 from their chromosomes, do not have this capability. These data support the hypothesis that phosphorylation of SYN1/SCP1 and COR1/SCP3 targets their removal from the chromosomes and that activity of the kinases involved correlates with the presence of these two proteins on the chromosomes.

Identification of the mouse beta'-COP Golgi component as a spermatocyte autoantigen in scleroderma and mapping of its gene Copb2 to mouse chromosome 9.

In an immunological screening of a mouse testicular cDNA library with a human CREST serum we isolated five overlapping cDNA clones encoding the mouse homolog of a Golgi coatomer complex protein (accession number AF043120), designated beta'-COP in bovine and p102 in humans. We generated antibodies against this protein which specifically recognize the Golgi apparatus of mouse spermatocytes. FISH analyses assigned the beta'-COP gene Copb2 to mouse Chromosome 9, region E3-F1. Our results demonstrate that CREST sera can contain antibody components against Golgi proteins as well as against nuclear proteins.

Chromosome cores and chromatin at meiotic prophase.

We review the synaptonemal complex, SC, of the synapsed homologous chromosomes at meiotic prophase in insects and mammals in terms of its formation, and the association of specific chromatin elements with the synaptonemal complexes. The focus is: (1) The SC as visualized with a variety of techniques; (2) The nature of the chromatin loops where they are associated with the SCs--the bases of the loops may be instrumental in recombinant events judging from the presence of Rad51 protein and late recombination nodules at the SCs; (3) Differences in DNA content of similarly sized loops; (4) Requirements for chromatin attachment to the chromosome cores, requirements that are apparently lacking in foreign DNA inserts; (5) Regulation of loop size by the position along the chromosome; (6) The structural correlates of recombination at the SCs--these comments are based on studies of SC structure, DNA-core protein associations, fluorescent in situ hybridization to visualize specific DNA segments, and fluorescent immunocytology to visualize the chromosome core proteins.

Protein coding gene trees in ciliates: comparison with rRNA-based phylogenies.

We have reexamined the phylogeny of the ciliates using alpha-tubulin and phosphoglycerate kinase gene sequences. For alpha-tubulin, we have compared the amino acid and nucleotide sequences of 20 species representing seven of the nine classes of the phylum (Karyorelictea, Heterotrichea, Hypotrichea, Oligohymenophorea, Colpodea, Nassophorea, and Litostomatea). The phylogenetic tree resembles a bush from which three monophyletic lineages can be distinguished which correspond to the three classes Hypotrichea, Oligohymenophorea, and Litostomatea. For phosphoglycerate kinase, we have compared the amino acid sequences from 7 species representing three classes (Heterotrichea, Hypotrichea, and Oligohymenophorea). The branching pattern is resolved in three deeply separated branches with an early emergence of the heterotrich. Our comparative analysis shows that if alpha-tubulin phylogeny is not informative at the interclass level, the preliminary data from the phosphoglycerate kinase molecule appear more promising. Nevertheless, at low taxonomic level and at the class level, the resolved phylogenetic relationships inferred from both protein and rRNA sequence data are congruent.

Protein-protein interactions in the synaptonemal complex.

In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II.

An unusual fibrillarin gene and protein: structure and functional implications.

The diploid germinal nucleus of the ciliated protozoan Tetrahymena thermophila is unusual among eukaryotes in that it encodes a single copy of the gene for rRNA allowing identification of cis-acting mutations in rDNA affecting rRNA structure, function, and processing. The generally conserved nucleolar protein fibrillarin has been characterized from a number of systems and is involved in pre-rRNA processing. We have demonstrated that Tetrahymena has fibrillarin and have analyzed the cDNA and the genomic DNA encoding this protein. The derived amino acid sequence of the N-terminal region of Tetrahymena fibrillarin shows little similarity with the generally highly conserved glycine/arginine-rich N-terminal domain of other eukaryotic fibrillarins. The remainder of the amino acid sequence of the molecule is more conserved. Polyclonal antibodies generated against the full-length Tetrahymena fibrillarin expressed in bacteria recognize a protein of M(r) approximately 32,000 in whole-cell or nucleolar preparations. Immunocytochemistry localizes fibrillarin to nucleoli in the somatic macronuclei of vegetative cells. Transformation experiments demonstrate that fibrillarin is an essential protein in Tetrahymena. The Tetrahymena fibrillarin is expressed but does not complement a NOP1 null mutation when transformed into the yeast Saccharomyces cerevisiae, indicating less functional conservation among fibrillarins than previously suggested.

Cloning, and molecular characterization of the GCV1 gene encoding the glycine cleavage T-protein from Saccharomyces cerevisiae.

We have isolated the gene encoding the glycine cleavage T-protein (GCV1) of the yeast Saccharomyces cerevisiae and shown through gene disruption and enzyme assays that inactivation of GCV1 destroys glycine cleavage function. A DNA fragment encoding the GCV1 gene was cloned by PCR amplification using degenerate oligodeoxyribonucleotides, and the cloned fragment was used as a probe to isolate the complete gene from a yeast genomic library. Growth with glycine stimulated expression of the GCV1 gene as determined by Northern analysis and increased the beta-galactosidase activity of a GCV1-lacZ fusion 30-fold. The URA3 gene was inserted into the coding sequence of GCV1 and the resulting construct was used to disrupt the chromosomal GCV1 gene in a diploid strain of yeast. gcv1::URA3 haploid derivatives grew normally or only slightly more slowly than the isogenic wild-type haploids. All gcv1 strains studied were unable to grow on glycine as a sole nitrogen source and lacked glycine cleavage enzyme activity. Growth of shm1 shm2 mutants was stimulated by glycine, whereas glycine could not supplement the growth of the isogenic gcv1 strain.

Programmed DNA rearrangement from an intron during nuclear development in Tetrahymena thermophila: molecular analysis and identification of potential cis-acting sequences.

During macronuclear development in the ciliate Tetrahymena thermophila, extensive rearrangement events occur as DNA deletions. We have studied a developmentally programmed deletion called mse2.9 that occurs within an intron in a gene in both genomic DNA and in an rDNA vector introduced into the cell by transformation. Extensive microheterogeneity at the deletion junctions has been found in caryonidal strains and in the rDNA in transformed cells. A transformation assay has been used to identify sequences required for proper processing of mse2.9. Models to explain deletion site selection as well as microheterogeneity at junction sites are presented.

In vivo analysis of folate coenzymes and their compartmentation in Saccharomyces cerevisiae.

In eukaryotes, enzymes responsible for the interconversion of one-carbon units exist in parallel in both mitochondria and the cytoplasm. Strains of Saccharomyces cerevisiae were constructed that possess combinations of gene disruptions at the SHM1 [mitochondrial serine hydroxymethyltransferase (SHMTm)], SHM2 [cytoplasmic SHMT (SHMTc)], MIS1 [mitochondrial C1-tetrahydrofolate synthase (C1-THFSm)], ADE3 [cytoplasmic C1-THF synthase (C1-THFSc)], GCV1 [glycine cleavage system (GCV) protein T], and the GLY1 (involved in glycine synthesis) loci. Analysis of the in vivo growth characteristics and phenotypes was used to determine the contribution to cytoplasmic nucleic acid and amino acid anabolism by the mitochondrial enzymes involved in the interconversion of folate coenzymes. The data indicate that mitochondria transport formate to the cytoplasmic compartment and mitochondrial synthesis of formate appears to rely primarily on SHMTm rather than the glycine cleavage system. The glycine cleavage system and SHMTm cooperate to specifically synthesize serine. With the inactivation of SHM1, however, the glycine cleavage system can make an observable contribution to the level of mitochondrial formate. Inactivation of SHM1, SHM2 and ADE3 is required to render yeast auxotrophic for TMP and methionine, suggesting that TMP synthesized in mitochondria may be available to the cytoplasmic compartment.

Genetic code deviations in the ciliates: evidence for multiple and independent events.

In several species of ciliates, the universal stop codons UAA and UAG are translated into glutamine, while in the euplotids, the glutamine codon usage is normal, but UGA appears to be translated as cysteine. Because the emerging position of this monophyletic group in the eukaryotic lineage is relatively late, this deviant genetic code represents a derived state of the universal code. The question is therefore raised as to how these changes arose within the evolutionary pathways of the phylum. Here, we have investigated the presence of stop codons in alpha tubulin and/or phosphoglycerate kinase gene coding sequences from diverse species of ciliates scattered over the phylogenetic tree constructed from 28S rRNA sequences. In our data set, when deviations occur they correspond to in frame UAA and UAG coding for glutamine. By combining these new data with those previously reported, we show that (i) utilization of UAA and UAG codons occurs to different extents between, but also within, the different classes of ciliates and (ii) the resulting phylogenetic pattern of deviations from the universal code cannot be accounted for by a scenario involving a single transition to the unusual code. Thus, contrary to expectations, deviations from the universal genetic code have arisen independently several times within the phylum.

MluI site-dependent transcriptional regulation of the Candida albicans dUTPase gene.

The Candida albicans dUTP pyrophosphatase (dUTPase) gene DUT1 has been isolated by genetic complementation in S. cerevisiae. It was found to encode a 17-kDa protein similar in amino-acid sequence to dUTPases isolated from other systems. The gene was adapted for expression in E. coli and yielded a soluble and highly-active enzyme which is easily purified. The 5' flanking sequence of DUT1 contains an MluI site typical of MCB cell-cycle-dependent UAS elements of budding and fission yeast. We found the gene to be cell-cycle-regulated when expressed in S. cerevisiae, and deletion of the MluI site resulted in a large reduction of DUT1 transcription in C. albicans. These results suggest that MCB elements are functionally conserved in this pathogenic fungus. Based on the vital role that dUTPase plays in DNA replication, the C. albicans enzyme may be a potentially useful target for the development of novel anti-fungal compounds.