Developmental distribution of the polyadenylation protein CstF-64 and the variant tauCstF-64 in mouse and rat testis

Messenger RNA polyadenylation is one of the processes that control gene expression in all eukaryotic cells and tissues. In mice, two forms of the regulatory polyadenylation protein CstF-64 are found. The gene Cstf2 on the X chromosome encodes this form, and it is expressed in all somatic tissues. The second form, tauCstF-64 (encoded by the autosomal gene Cstf2t), is expressed in a more limited set of tissues and cell types, largely in meiotic and postmeiotic male germ cells and, to a smaller extent, in brain. We report here that whereas CstF-64 and tauCstF-64 expression in rat tissues resembles their expression in mouse tissues, significant differences also are found. First, unlike in mice, in which CstF-64 was expressed in postmeiotic round and elongating spermatids, rat CstF-64 was absent in those cell types. Second, unlike in mice, tauCstF-64 was expressed at significant levels in rat liver. These differences in expression suggest interesting differences in X-chromosomal gene expression between these two rodent species.     

The gene for a variant form of the polyadenylation protein CstF-64 is on chromosome 19 and is expressed in pachytene spermatocytes in mice

Many mRNAs in male germ cells lack the canonical AAUAAA but are normally polyadenylated (Wallace, A. M., Dass, B., Ravnik, S. E., Tonk, V., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and MacDonald, C. C. (1999) Proc. Natl. Acad Sci. U. S. A. 96, 6763-6768). Previously, we demonstrated the presence of two distinct forms of the M(r) 64,000 protein of the cleavage stimulation factor (CstF-64) in mouse male germ cells and in brain, a somatic M(r) 64,000 form and a variant M(r) 70,000 form. The variant form was specific to meiotic and postmeiotic germ cells. We localized the gene for the somatic CstF-64 to the X chromosome, which would be inactivated during male meiosis. This suggested that the variant CstF-64 was an autosomal homolog activated during that time. We have named the variant form "tau CstF-64," and we describe here the cloning and characterization of the mouse tauCstF-64 cDNA, which maps to chromosome 19. The mouse tauCstF-64 protein fits the criteria of the variant CstF-64, including antibody reactivity, size, germ cell expression, and a common proteolytic digest pattern with tauCstF-64 from testis. Features of mtauCstF-64 that might allow it to promote the germ cell pattern of polyadenylation include a Pro --> Ser substitution in the RNA-binding domain and significant changes in the region that interacts with CstF-77.     

Polymorphisms of the human PUMILIO2 gene and male sterility

The highly conserved Pumilio protein plays crucial roles in fertility of many organisms acting as a repressor of translation, and causing infertility when mutated. Although one of two human Pumilio homologs, PUMILIO2 is expressed mainly in the germ line, its role in mammalian germ cell development has not been reported yet. To shed light on the role of PUMILIO2 in development of the human male germ line, we screened this gene for mutations in 137 patients presenting a variety of phenotypes with spermatogenic failure. The first variant, we identified was a single base substitution within intron 15 (IVS15 + 6G > A). This variant was found in three azoospermic males, the second allele being the wild type. However, this variant was also present among fertile males, as frequently as in the patients. Although location of IVS15 + 6G > A substitution in close proximity to the canonical donor splice site GT, indicates that its influence on splicing cannot be excluded, our preliminary cDNA analysis has not revealed evidence of a splicing abnormality of PUMILIO2 pre-mRNA carrying this variant. Nevertheless, this study provides new interesting variant containing a donor splice site variant, which can be relevant for understanding of splicing mechanism of mammalian genes. The second variant, c.774 C > T transversion (Y258Y) in exon 6 was found only in one patient, but an influence on PUMILIO2 function is not obvious. Altogether, this study shows that variation in the PUMILIO2 gene is very low and it seems improbable that mutations of this gene significantly contribute to male infertility in humans.     

A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein

We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3' UTR) in HPV-16 gene expression. We found that deletion of the early 3' UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3' half of the early 3' UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3' splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.     

The gene encoding the nonstructural protein of B19 (human) parvovirus may be lethal in transfected cells.

The B19 parvovirus is a cause of bone marrow failure in humans. B19 is toxic to erythroid progenitor cells in vitro. Viral products possibly responsible for toxicity were explored by transfection of cloned B19 genome into HeLa cells. The nonstructural (NS) protein was detected in cells 30 h after transfection. Plasmids containing the B19 genome were transfected with selectable marker genes in stable transformation assays. Plasmids that contained the left side of the B19 genome, which encodes the NS protein of the virus, inhibited antibiotic-resistant colony formation. Transformation occurred when NS protein expression was blocked by mutation. Suppression of transformation by NS protein was not tissue specific, suggesting a role for NS protein in toxicity for nonpermissive cells without parvovirus replication or virion accumulation.     

DCW11, down-regulated gene 11 in CW-type cytoplasmic male sterile rice, encoding mitochondrial protein phosphatase 2c is related to cytoplasmic male sterility

Causes of cytoplasmic male sterility (CMS) in plants have beenstudied for two decades, and mitochondrial chimeric genes havebeen predicted to induce CMS. However, it is unclear what happensafter CMS-associated proteins accumulate in mitochondria. Inour previous study of microarray analysis, we found that 140genes are aberrantly regulated in anthers of CW-type CMS ofrice (Oryza sativa L.). In the present study, we investigatedDCW11, one of the down-regulated genes in CW-CMS encoding aprotein phosphatase 2C (PP2C). DCW11 mRNA was preferentiallyexpressed in anthers, with the highest expression in maturepollen. As predicted by the N-terminal sequence, DCW11 signalpeptide–green fluorescent protein (GFP) fusion proteinwas localized in mitochondria. Knockdown of DCW11 in wild-typerice by RNA interference caused a major loss of seed-set fertility,without visible defect in pollen development. Since this knockdownphenotype resembled that of CW-CMS, we concluded that the down-regulationof DCW11 is correlated with CW-CMS. This idea was supportedby the up-regulation of alternative oxidase 1a (AOX1a), whichis known to be regulated by mitochondrial retrograde signaling,in DCW11 knockdown lines. Down-regulation of DCW11 and up-regulationof AOX1a were also observed in two other types of rice CMS.Our result indicates that DCW11 could play a role as a mitochondrialsignal transduction mediator in pollen germination.     

The reovirus M1 gene, encoding a viral core protein, is associated with the myocarditic phenotype of a reovirus variant.

Reoviruses contain a genome composed of 10 double-stranded RNA gene segments. A reovirus reassortant, 8B, derived from type 1 Lang (T1L) and type 3 Dearing (T3D), displayed a phenotype unlike that of either of its parents in that it efficiently induced numerous macroscopic external cardiac lesions in neonatal mice (B. Sherry, F. J. Schoen, E. Wenske, and B. N. Fields, J. Virol. 63:4840-4849, 1989). A panel of T1L/T3D reassortants and a panel of reassortants derived from 8B were used to determine whether novel T1L/T3D gene associations in 8B were responsible for its myocarditic phenotype. The results eliminated the possibility that any T1L/T3D gene combination found in 8B, from 2 genes to all 10 genes, was the explanation for its induction of cardiac lesions. This suggested that a mutation(s) in an 8B gene(s) might be responsible for induction of the myocarditis. Statistical analysis of experiments with 31 reassortants derived from 8B revealed a highly significant association (P = 0.002) of the 8B M1 gene with induction of cardiac lesions. The reovirus M1 gene encodes a viral core protein of unknown function, although evidence suggests a potential role in core structure and/or viral RNA synthesis. This represents the first report of the association of a viral gene with induction of myocarditis.     

Structure, mapping and expression of the human gene encoding the homeodomain protein, SIX2

Vertebrate genes with sequence similarity to the Drosophila homeobox gene, sine oculis (so), constitute the SIX family. There is notable expression of members of this family in anterior neural structures, and several SIX genes have been shown to play roles in vertebrate and insect development, or have been implicated in maintenance of the differentiated state of tissues. Mutations in three of these genes in man (SIX5, SIX6 and SIX3) are associated with severe phenotypes, and therefore, the cloning of other human genes from this family is of interest. We have cloned and characterised the gene that encodes human SIX2, elucidated its gene structure and conducted expression studies in a range of tissues. SIX2 is widely expressed in the late first-trimester fetus, but has a limited range of expression sites in the adult. The expression pattern of SIX2 and its localisation to chromosome 2p15-p16 will be of use in assessing its candidacy in human developmental disorders.     

The fertility restorer gene, Rf2, for Lead Rice-type cytoplasmic male sterility of rice encodes a mitochondrial glycine-rich protein

Cytoplasmic male sterility (CMS) is associated with a mitochondrial mutation that causes an inability to produce fertile pollen. The fertility of CMS plants is restored in the presence of a nuclear-encoded fertility restorer (Rf) gene. In Lead Rice-type CMS, discovered in the indica variety 'Lead Rice', fertility of the CMS plant is restored by the single nuclear-encoded gene Rf2 in a gametophytic manner. We performed map-based cloning of Rf2, and proved that it encodes a protein consisting of 152 amino acids with a glycine-rich domain. Expression of Rf2 mRNA was detected in developing and mature anthers. An RF2-GFP fusion was shown to be targeted to mitochondria. Replacement of isoleucine by threonine at amino acid 78 of the RF2 protein was considered to be the cause of functional loss in the rf2 allele. As Rf2 does not encode a pentatricopeptide repeat protein, unlike a majority of previously identified Rf genes, the data from this study provide new insights into the mechanism for restoring fertility in CMS.     

Cloning, characterization, and mapping of the gene encoding the human G protein gamma 2 subunit

G proteins play vital roles in cellular responses to external signals. The specificity of G protein-receptor interaction is mediated mostly by the gamma-subunit and the individual members of the gamma-subunit multigene family would hence be expected to each have a particular expression profile. In an experiment designed to isolate genes expressed predominantly in human testis we identified a cDNA fragment corresponding to the gamma2 gene. Although the protein sequence of the gamma2 subunit has previously been published, the cDNA sequence, expression pattern, genomic structure, and localisation of the human GNG2 gene have not been described. We report the complete sequence of the GNG2 cDNA which is 1066 bp long and contains an open reading frame encoding a protein of 71 amino acids. This protein is 100% homologous to the bovine, mouse, and rat G protein gamma2 subunit. The gene structure is very similar to that of other Ggamma-subunit genes in that there are two introns, one located in the 5' UTR and the other within the ORF. We show that this gene is expressed in a range of foetal tissues as well as adult testis, adrenal gland, brain, white blood cells and lung but not in adult liver, muscle, sperm, prostate gland nor in the testes of two different infertile patients. There is evidence that GNG2 is expressed in malignant tissues. Using two independent methods, we have mapped the human GNG2 gene to chromosome 14q21.     

TPH gene may be associated with suicidal behavior, but not with schizophrenia in the Korean population

Tryptophan hydroxylase (TPH) is a rate-limiting enzyme in the biosynthesis of serotonin. This study was designed to examine whether A218C polymorphism, which has been identified in intron 7 of the TPH gene, may be associated with schizophrenia or the suicidal behavior of schizophrenics in the Korean population. TPH genotypes were determined in DNA samples from 217 schizophrenics and 236 healthy volunteers. Among the schizophrenic group, 27 patients had a history of suicidal behavior. Genomic DNA was amplified by a polymerase-chain-reaction-based method and restricted by Nhel. A218C polymorphism was associated with a history of suicidal behavior in schizophrenics. This finding suggests that the TPH gene or a gene in its vicinity may influence suicidal behavior in schizophrenics. However, genotypic and allelic distrubutions of this polymorphism did not significantly differ between schizophrenics and controls.     

The human thyroglobulin gene is over 300 kb long and contains introns of up to 64 kb.

Thyroglobulin (Tg), the precursor of thyroid hormones, is a 660.000 Da dimeric glycoprotein synthesized exclusively in the thyroid gland. We have cloned the human thyroglobulin gene from cosmid and phage libraries and constructed a complete restriction map. The gene encodes an 8.7 kb mRNA, covers at least 300 kb DNA and contains at least 37 exons separated by large introns of up to 64 kb. A striking difference in structure between the 5' and 3' part of the gene suggests that it is composed of two evolutionarily different regions. The first 30 kb DNA encode 3 kb of the mRNA, yielding an exon:intron ratio of 1:10, whereas the remaining 270 kb encodes 5.7 kb of the mRNA with an exon:intron ratio of 1:47. In thyroid cells, the Tg gene is not rearranged and nuclear RNA homologous with sequences internal to the 64 kb intron is present, suggesting that the Tg gene is transcribed as a 300 kb RNA.     

cDNA cloning, characterization and chromosome mapping of the gene encoding human cartilage associated protein (CRTAP)

We have recently isolated and characterized cDNA clones coding for a novel developmentally regulated avian and mouse embryo protein, CASP for Cartilage Associated Protein. Here we describe the isolation and characterization of the gene coding for the human CASP. The comparison of the putative human and mouse protein sequences with the chick sequence revealed an overall high identity (89% and 51%, respectively). Homology search with known DNA and protein sequences showed that CASPs are related to two mammalian nuclear proteins. Here we demonstrate definitively that CASPs are distinct from these nuclear proteins. However, sequence comparison analyses suggest that all of these proteins belong to a new family. In all human tissues examined two CASP mRNA species were detected, whereas a single mRNA and three mRNAs were found in chick and mouse, respectively. The human CASP gene (CRTAP) was assigned to chromosome 3p22 by fluorescence in situ hybridization.