Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation

Germ-line mutation induction at mouse minisatellite loci by acute irradiation with x-rays was studied at premeiotic and postmeiotic stages of spermatogenesis. An elevated paternal mutation rate was found after irradiation of premeiotic spermatogonia and stem cells, whereas the frequency of minisatellite mutation after postmeiotic irradiation of spermatids was similar to that in control litters. In contrast, paternal irradiation did not affect the maternal mutation rate. A linear dose–response curve for paternal mutation induced at premeiotic stages was found, with a doubling dose of 0.33 Gy, a value close to those obtained in mice after acute spermatogonia irradiation using other systems for mutation detection. High frequencies of spontaneous and induced mutations at minisatellite loci allow mutation induction to be evaluated at low doses of exposure in very small population samples, which currently makes minisatellite DNA the most powerful tool for monitoring radiation-induced germ-line mutation.

A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice

Tuberous sclerosis (TS) is characterized by the development of hamartomas in various organs and is caused by a germ-line mutation in either TSC1 or TSC2 tumor suppressor genes. From the symptomatic resemblance among TS patients, involvement of TSC1 and TSC2 products in a common pathway has been suggested. Here, to analyze the function of the Tsc1 product, we established a line of Tsc1 (TSC1 homologue) knockout mouse by gene targeting. Heterozygous Tsc1 mutant (Tsc1+/−) mice developed renal and extra-renal tumors such as hepatic hemangiomas. In these tumors, loss of wild-type Tsc1 allele was observed. Homozygous Tsc1 mutants died around embryonic days 10.5–11.5, frequently associated with neural tube unclosure. As a whole, phenotypes of Tsc1 knockout mice resembled those of Tsc2 knockout mice previously reported, suggesting that the presumptive common pathway for Tsc1 and Tsc2 products may also exist in mice. Notably, however, development of renal tumors in Tsc1+/− mice was apparently slower than that in Tsc2+/− mice. The Tsc1 knockout mouse described here will be a useful model to elucidate the function of Tsc1 and Tsc2 products as well as pathogenesis of TS.

Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma:analysis of a clinic-based population

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.

Production of zebrafish germ-line chimeras from embryo cell cultures

Although the zebrafish possesses many characteristics that make it a valuable model for genetic studies of vertebrate development, one deficiency of this model system is the absence of methods for cell-mediated gene transfer and targeted gene inactivation. In mice, embryonic stem cell cultures are routinely used for gene transfer and provide the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. Transgenic animals possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of a chimeric embryo. Although zebrafish embryo cell cultures that exhibit characteristics of embryonic stem cells have been described, successful contribution of the cells to the germ-cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures maintained in the presence of cells from a rainbow trout spleen cell line (RTS34st) are able to produce germ-line chimeras when introduced into a host embryo. Messenger RNA encoding the primordial germ-cell marker, vasa, was present for more than 30 days in embryo cells cocultured with RTS34st cells or their conditioned medium and disappeared by 5 days in the absence of the spleen cells. The RTS34st cells also inhibited melanocyte and neuronal cell differentiation in the embryo cell cultures. These results suggest that the RTS34st splenic–stromal cell line will be a valuable tool in the development of a cell-based gene transfer approach to targeted gene inactivation in zebrafish.

CDKN2A mutation in a non-FAMMM kindred with cancers at multiple sites results in a functionally abnormal protein

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.

Increased concentrations of the oxidative DNA adduct 7, 8-dihydro-8-oxo-2-deoxyguanosine in the germ-line of men with type 1 diabetes

The effects of diabetes mellitus on male reproductive health have not been clearly defined. A previous publication from this group reported significantly higher levels of nuclear DNA fragmentation and mitochondrial DNA deletions in spermatozoa from men with type 1 diabetes. This study compared semen profiles, sperm DNA fragmentation and levels of oxidative DNA modification in spermatozoa of diabetic and non-diabetic men. Semen samples from 12 non-diabetic, fertile men and 11 type 1 diabetics were obtained and subjected to conventional light microscopic semen analysis. Nuclear DNA fragmentation was assessed using an alkaline Comet assay and concentrations of 7,8-dihydro-8-oxo-2-deoxyguanosine (8-OHdG), an oxidative adduct of the purine guanosine, were assessed by high-performance liquid chromatography. Conventional semen profiles were similar in both groups, whilst spermatozoa from type 1 diabetics showed significantly higher levels of DNA fragmentation (44% versus 27%; P < 0.05) and concentrations of 8-OHdG (3.6 versus 2.0 molecules of 8-OHdG per 105 molecules of deoxyguanosine; P < 0.05). Furthermore, a positive correlation was observed between DNA fragmentation and concentrations of 8-OHdG per 105 molecules of deoxyguanosine (rs = 0.7, P < 0.05). The genomic damage evident in spermatozoa of type 1 diabetics may have important implications for their fertility and the outcome of pregnancies fathered by these individuals.

DNA methyltransferase expression in the mouse germ line during periods of de novo methylation

DNA methyltransferase (DNMT) 3A and DNMT3B are both active de novo DNA methyltransferases required for development, whereas DNMT3L, which has no demonstrable methyltransferase activity, is required for methylation of imprinted genes in the oocyte. We show here that different mechanisms are used to restrict access by these proteins to their targets during germ cell development. Transcriptional control of the Dnmt3l promoter guarantees that message is low or absent except during periods of de novo activity. Use of an alternative promoter at the Dnmt3a locus produces the shorter Dnmt3a2 transcript in the germ line and postimplantation embryo only, whereas alternative splicing of the Dnmt3b transcript ensures that Dnmt3b1 is absent in the male prospermatogonia. Control of subcellular protein localization is a common theme for DNMT3A and DNMT3B, as proteins were seen in the nucleus only when methylation was occurring. These mechanisms converge to ensure that the only time that functional products from each locus are present in the germ cell nuclei is around embryonic day 17.5 in males and after birth in the growing oocytes in females.

Germ-line chimaeras can produce both strains of fowl with high efficiency after partial sterilization

The drug busulphan is known to be cytotoxic to migrating primordial germ cells (PGCs). A technique is described in which doses of 0, 25, 50 and 250 micrograms busulphan in 40 microliters sesame oil were injected into the yolk of White Leghorn eggs incubated for 0, 24, 48 and 72 h. The percentage survival values of these embryos showed that the older the embryo at the time of injection, the greater the survival. Increasing the dose of busulphan decreased the survival. The percentage of embryos showing abnormalities increased with higher doses of busulphan. The number of germ cells in histological sections from gonads of 16-day embryos was estimated and in embryos treated with 50 micrograms and 250 micrograms busulphan the number of germ cells was significantly less than in the controls. Eggs were injected with 50 micrograms busulphan at 24-30 h, and at 50-55 h the embryos received an intravascular injection of a germinal crescent cell suspension containing PGCs from Rhode Island Red embryos. Twenty hatchlings from these experiments were raised to sexual maturity. All these birds were fertile and half of the breeding groups producing offspring from the transferred germ cells at a rate of about 35% of the total. The technique would improve the efficiency of producing transgenic gametes.

Pronounced Segregation of Donor Mitochondria Introduced by Bovine Ooplasmic Transfer to the Female Germ-Line

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Germ line Cysts and the Formation of the Germinal Epithelium During the Female Gonadal Morphogenesis in Cyprinus Carpio (Teleostei: Ostariophysi: Cypriniformes)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mitochondrial inheritance and germ line determination in a bivalve species with Doubly Uniparental Inheritance (DUI)

Mitochondria are inherited maternally in most metazoans. However, in some bivalves, two mitochondrial lineages are present: one transmitted through eggs (F), the other through sperm (M). This is called Doubly Uniparental Inheritance (DUI). During male embryo development, spermatozoon mitochondria aggregate and end up in the primordial germ cells, while they are dispersed in female embryos. The molecular mechanisms of segregation patterns are still unknown. In the DUI species Ruditapes philippinarum, I examined sperm mitochondria distribution by MitoTracker, microtubule staining and TEM, and I localized germ line determinants with immunocytochemical analysis. I also analyzed the gonad transcriptome, searching for genes involved in reproduction and sex determination. Moreover, I analyzed an M-type specific open reading frame that could be responsible for maintenance/degradation of M mitochondria during embryo development. These transcripts were also localized in tissues using in situ hybridization. As in Mytilus, two distribution patterns of M mitochondria were detected in R. philippinarum, supporting that they are related to DUI. Moreover, the first division midbody concurs in positioning aggregated M mitochondria on the animal-vegetal axis of the male embryo: in organisms with spiral segmentation this zone is not involved in further cleavages, so aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area where germ plasm is transferred, suggesting their contribution in male germ line formation. The finding of reproduction and ubiquitination transcripts led to formulate a model in which ubiquitination genes stored in female oocytes during gametogenesis would activate sex-gene expression in the early embryonic developmental stages (preformation). Only gametogenetic cells were labeled by in situ hybridization, proving their specific transcription in developing gametes. Other than having a role in sex determination, some ubiquination factors could also be involved in mitochondrial inheritance, and their differential expression could be responsible for the different fate of sperm mitochondria in the two sexes.

Role of germ line p53 mutations in aneuploidy and immortalization of dermal fibroblasts from Li-Fraumeni cancer patients

Pedigree analysis of certain families with a high incidence of tumors suggests a genetic predisposition to cancer. Li and Fraumeni described a familial cancer syndrome that is characterized by multiple primary tumors, early age of onset, and marked variation in tumor type. Williams and Strong (1) demonstrated that at least 7% of childhood soft tissue sarcoma patients had family histories that is readily explained by a highly penetrant autosomal dominant gene. To characterize the mechanism for genetic predisposition to many tumor types in these families, we have studied genetic alterations in fibroblasts, a target tissue from patients with the Li-Fraumeni Syndrome (LFS).^ We have observed spontaneous changes in initially normal dermal fibroblasts from LFS patients as they are cultured in vitro. The cells acquire an altered morphology, chromosomal anomalies, and anchorage-independent growth. This aberrant behavior of fibroblasts from LFS patients had never been observed in fibroblasts from normal donors. In addition to these phenotypic alterations, patient fibroblasts spontaneously immortalize by 50 population doublings (pd) in culture; unlike controls that remain normal and senesce by 30-35 (2). At 50 pd, immortal fibroblasts from two patients were found to be susceptible to tumorigenic transformation by an activated T24 H-ras oncogene (3). Approximately 80% of the oncogene expressing transfectants were capable of forming tumors in nude mice within 2-3 weeks. p53 has been previously associated with immortalization of cells in culture and cooperation with ras in transfection assays. Therefore, patients' fibroblast and lymphocyte derived DNA was tested for point mutations in p53. It was shown that LFS patients inherited certain point mutations in one of the two p53 alleles (4). Further studies on the above LFS immortal fibroblasts have demonstrated loss of the remaining p53 allele concomitant with escape from senescence. While the loss of the second allele correlates with immortalization it is not sufficient to transformation by an activated H-ras or N-ras oncogene. These immortal fibroblasts are resistant to tumorigenic transformation by v-abl, v-src, c-neu or v-mos oncogene; implying that additional steps are required in the tumorigenic progression of LFS patients' fibroblasts.^ References. (1) Williams et al., J. Natl. Cancer Inst. 79:1213, 1987. (2) Bischoff et al., Cancer Res. 50:7979, 1990. (3) Bischoff et al., Oncogene 6:183, 1991. (4) Malkin et al., Science 250:1233, 1990. ^

GENETIC INTERACTIONS OF FACTORS THAT REGULATE ALTERNATIVE RNA SPLICING IN THE MALE GERM LINE OF DROSOPHILA

Alternative RNA splicing is a critical process that contributes variety to protein functions, and further controls cell differentiation and normal development. Although it is known that most eukaryotic genes produce multiple transcripts in which splice site selection is regulated, how RNA binding proteins cooperate to activate and repress specific splice sites is still poorly understood. In addition how the regulation of alternative splicing affects germ cell development is also not well known. In this study, Drosophila Transformer 2 (Tra2) was used as a model to explore both the mechanism of its repressive function on its own pre-mRNA splicing, and the effect of the splicing regulation on spermatogenesis in testis. Half-pint (Hfp), a protein known as splicing activator, was identified in an S2 cell-based RNAi screen as a co-repressor that functions in combination with Tra2 in the splicing repression of the M1 intron. Its repressive splicing function is found to be sequence specific and is dependent on both the weak 3’ splice site and an intronic splicing silencer within the M1 intron. In addition we found that in vivo, two forms of Hfp are expressed in a cell type specific manner. These alternative forms differ at their amino terminus affecting the presence of a region with four RS dipeptides. Using assays in Drosophila S2 cells, we determined that the alternative N terminal domain is necessary in repression. This difference is probably due to differential localization of the two isoforms in the nucleus and cytoplasm. Our in vivo studies show that both Hfp and Tra2 are required for normal spermatogenesis and cooperate in repression of M1 splicing in spermatocytes. But interestingly, Tra2 and Hfp antagonize each other’s function in regulating germline specific alternative splicing of Taf1 (TBP associated factor 1). Genetic and cytological studies showed that mutants of Hfp and Taf1 both cause similar defects in meiosis and spermatogenesis. These results suggest Hfp regulates normal spermatogenesis partially through the regulation of taf1 splicing. These observations indicate that Hfp regulates tra2 and taf1 activity and play an important role in germ cell differentiation of male flies.