Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa.

There is growing concern over the effect of sperm cryopreservation on DNA integrity and the subsequent development of offspring generated from this cryopreserved material. In the present study, membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation, a process that happens upon exposure to water to spermatozoa of some aquatic species. A dye exclusion assay revealed that sperm plasma membrane integrity in both species decreased after freezing, more so for X. laevis than X. tropicalis spermatozoa. The sperm chromatin dispersion (SCD) test showed that for both X. tropicalis and X. laevis, activated frozen spermatozoa produced the highest levels of DNA fragmentation compared with all fresh samples and frozen non-activated samples (P<0.05). Understanding the nature of DNA and membrane damage that occurs in cryopreserved spermatozoa from Xenopus species represents the first step in exploiting these powerful model organisms to understand the developmental consequences of fertilising with cryopreservation-damaged spermatozoa.

The maternal CCAAT box transcription factor which controls GATA-2 expression is novel and developmentally regulated and contains a double-stranded-RNA-binding subunit.

The transcription factor GATA-2 is expressed at high levels in the nonneural ectoderm of the Xenopus embryo at neurula stages, with lower amounts of RNA present in the ventral mesoderm and endoderm. The promoter of the GATA-2 gene contains an inverted CCAAT box conserved among Xenopus laevis, humans, chickens, and mice. We have shown that this sequence is essential for GATA-2 transcription during early development and that the factor binding it is maternal. The DNA-binding activity of this factor is detectable in nuclei and chromatin bound only when zygotic GATA-2 transcription starts. Here we report the characterization of this factor, which we call CBTF (CCAAT box transcription factor). CBTF activity mainly appears late in oogenesis, when it is nuclear, and the complex has multiple subunits. We have identified one subunit of the factor as p122, a Xenopus double-stranded-RNA-binding protein. The p122 protein is perinuclear during early embryonic development but moves from the cytoplasm into the nuclei of embryonic cells at stage 9, prior to the detection of CBTF activity in the nucleus. Thus, the accumulation of CBTF activity in the nucleus is a multistep process. We show that the p122 protein is expressed mainly in the ectoderm. Expression of p122 mRNA is more restricted, mainly to the anterior ectoderm and mesoderm and to the neural tube. Two properties of CBTF, its dual role and its cytoplasm-to-nucleus translocation, are shared with other vertebrate maternal transcription factors and may be general properties of these proteins.

Methods for the analysis of DNA-protein interactions.

The interaction of proteins with DNA is a central theme of molecular biology. In this article, we review some of the principal techniques currently used for the identification and characterization of DNA binding proteins, and for investigation of the molecular interactions that are responsible for the recognition of specific DNA sequences.

The detection of melanoma cells in peripheral blood by reverse transcription-polymerase chain reaction.

Both cutaneous and uveal melanoma undergo haematogenous dissemination. Detection of tyrosinase mRNA by reverse transcription-polymerase chain reaction (RT-PCR) has been described as an extremely sensitive way of detecting circulating viable melanoma cells in the peripheral venous blood, and this technique may be of value in the early detection of dissemination. Also, it has been suggested that surgical manipulation of the eye, such as occurs during enucleation, can provoke uveal melanoma dissemination. The purpose of this study was to evaluate whether tyrosinase mRNA is detectable in the peripheral blood of patients with uveal and cutaneous melanoma and in patients with uveal melanoma undergoing surgical procedures on the eye harbouring the tumour. Venous blood samples from 36 patients diagnosed as having active uveal melanoma and from six patients with advanced metastatic cutaneous melanoma were analysed. In addition, blood samples were spiked with known numbers of cells from three cell lines and four primary uveal melanoma cultures. The reported sensitivity of the technique was confirmed, with an ability to detect down to one cell per ml of blood. All 51 blood samples from the 36 patients with uveal melanoma were negative, and this included 20 perioperative blood samples. The test was also negative for the six patients with advanced cutaneous melanoma. There were two positives among 31 control samples analysed. This study demonstrates that there are far fewer circulating viable melanocytes than has been previously supposed in patients with melanoma and that the RT-PCR is of no clinical value in detecting metastatic melanoma disease. There was no evidence for surgery causing a bolus of melanoma cells to enter the peripheral circulation.

Estrogen and progesterone receptor analysis in ocular melanomas.

BACKGROUND: It has been suggested that pregnancy may promote metastases in melanoma and that the contraceptive pill may be an etiologic factor. The purpose of this study is to determine if uveal or conjunctival melanomas express estrogen or progesterone receptors. METHODS: Twenty-seven choroidal and five conjunctival melanomas were investigated. Immunohistochemistry was performed using the antibodies ER-D5, which recognizes heat-shock protein 27 (formerly called the estrogen receptor-associated cytoplasmic antigen); ER-1D5, which recognizes the estrogen receptor; and PgR, which recognizes the progesterone receptor. RESULTS: Most of the conjunctival and uveal melanomas stained strongly for heat-shock protein 27 but none of the tumors showed positive nuclear staining for either the estrogen or the progesterone receptor. CONCLUSIONS: No evidence was found for either estrogen receptor or progesterone receptor expression in choroidal or conjunctival melanomas. Based on the literature, there is little evidence for these hormones having a role in the development or progression of these tumors.

Nuclear translocation of a maternal CCAAT factor at the start of gastrulation activates Xenopus GATA-2 transcription.

The transcription factor GATA-2 is present in blood cell precursors and plays a pivotal role in the control of erythroid differentiation. In Xenopus embryos, low levels of GATA-2 mRNA are maternally derived, while the onset of zygotic GATA-2 expression coincides with commitment to haematopoietic lineages. However, its initial transcriptional activation is not restricted to the presumptive blood islands, but occurs throughout ventral and lateral regions, in all three germ layers. In order to determine how this expression pattern is controlled, we have isolated and characterized the Xenopus GATA-2 gene. We show that 1.65 kb of 5' flanking sequences are sufficient to direct both correct transcriptional initiation in oocytes and appropriate temporal and spatial gene expression in early embryos. The transgene is activated during gastrulation and by neurula stages in predominantly expressed in the ventral hemisphere. We demonstrate that a CCAAT element is necessary for gene activity in both systems and that extracts prepared from oocytes and embryos contain a factor which specifically recognizes this element. We also show that cytoplasmic localization inhibits the function of this CCAAT factor until the beginning of gastrulation, when the zygotic GATA-2 gene is activated. These observations extend our understanding of the mechanisms by which maternal factors control the temporal activation of transcription in early vertebrate embryos.

Negative control of Xenopus GATA-2 by activin and noggin with eventual expression in precursors of the ventral blood islands.

To increase our understanding of haematopoiesis during early vertebrate development, we have studied the expression pattern of the transcription factor GATA-2 in Xenopus embryos, and asked how this is regulated. We show that the blood island precursors of the ventral mesoderm express GATA-2 RNA at neural tube stages, some 5 hours before globin RNA is detected in their derivatives. Prior to this however, GATA-2 is expressed much more widely within the embryo. Maternal transcripts are uniformly distributed, and zygotic transcription is activated during gastrulation throughout ventral and lateral regions of the embryo, with expression highest in the sensorial ectoderm and only weak in the ventral mesoderm. The domain of GATA-2 expression in neurulae outlines the region of the neural plate and suggests a possible wider role in dorsoventral patterning. To identify the signals involved in regulating this pattern of expression, we performed experiments with embryo explants. GATA-2 is activated autonomously in isolated animal caps and this activation is suppressed by the mesoderm-inducing factor activin, but not by FGF. Thus, the down-regulation of GATA-2 observed in the region of the Spemann organiser may be a response to an activin-like signal emanating from the dorsal-vegetal region or Nieuwkoop centre. GATA-2 activation in animal caps and ventral marginal zones was suppressed by co-culturing with dorsal marginal zones, suggesting that a signal from the Spemann organiser is involved in suppression of GATA-2 in the dorsal region of the embryo. Expression of a candidate for this signal, noggin, had the same effect. Taken together, the observations presented here suggest that GATA-2 activation occurs by default in the absence of signals, that the restriction of its expression within the early embryo is controlled by negative signals emanating from the Nieuwkoop centre and the organiser, and that noggin and activin-like molecules play a role in these signalling pathways.

Functional differences in the promoters of the interferon-inducible (2'-5')A oligoadenylate synthetase and 6-16 genes in interferon-resistant Daudi cells.

A clone of interferon-alpha-resistant (IFNr) Daudi cells retained much greater transcriptional inducibility of the (2'-5') oligoadenylate synthetase than the 6-16 gene despite the fact that the response of both genes is mediated by highly similar interferon-stimulable DNA response elements (ISRE). The primary IFN-alpha activatable transcription factor E (ISGF3) and the additional IFN-alpha-inducible ISRE-binding complex M were greatly reduced in the IFNr cells. The defect in E was in the E alpha subunit. In electrophoretic mobility-shift assays the 6-16 and (2'-5') oligoadenylate synthetase ISRE competed approximately equivalently for E and M. Moreover although active in wild-type cells the (2'-5') oligoadenylate synthetase ISRE was no more capable of conferring inducibility on a reporter gene in the IFNr cells than was the 6-16 ISRE. The contrasting response of the endogenous (2'-5') oligoadenylate synthetase and 6-16 genes in the IFNr cells is, therefore, unlikely simply to reflect the slight difference in the sequence of their ISRE. Consistent with this, in addition to the ISRE, sequences 5' to the ISRE in the (2'-5') oligoadenylate synthetase promoter appeared necessary for good induction by IFN alpha in the IFNr cells. Subtle quantitative changes in the phenotype of the IFNr cells have, however, precluded a more precise definition of the DNA element(s) involved.

Overlapping sites for constitutive and induced DNA binding factors involved in interferon-stimulated transcription.

A 14 bp interferon (IFN)-stimulated response element (ISRE) from 6-16, a human gene regulated by alpha-IFN, confers IFN inducibility on a heterologous thymidine kinase promoter. A 39 bp double-stranded oligonucleotide corresponding to a 5' region of 6-16 which includes the ISRE competes for factors required for gene expression by alpha-IFN in transfected cells and a single base change (A-11 to C) within the ISRE (GGGAAAATGAAACT) abolishes this competition. Band-shift assays performed with whole-cell extracts and the 39 bp oligonucleotide reveal specific complexes formed by rapidly induced and constitutive factors, both of which fail to bind to the A-11 to C oligonucleotide. A detailed footprinting analysis reveals that these two types of factors bind to overlapping sites within the ISRE, but in very different ways. These data were used to design oligonucleotides which decreased the formation of the inducible complex without affecting the constitutive one. Changes at the 5' margin of the ISRE and upstream of it markedly decrease formation of the induced but not the constitutive complex and also abolish the ability of the 39 bp sequence to function as an inducible enhancer with the thymidine kinase promoter. Thus, induction of 6-16 transcription in IFN-treated cells is likely to be stimulated by binding of the induced factor to the ISRE and upstream sequences, while the subsequent suppression of transcription may involve competition for the ISRE by the other class of factors.

The anti-oestrogen drug tamoxifen is an elongation inhibitor of eukaryotic protein biosynthesis.

The drug tamoxifen is widely used in the chemotherapy of breast cancer but its action is not explained completely by its anti-oestrogen properties. We now present evidence indicating that it is also a potent inhibitor of eukaryotic protein synthesis as demonstrated in Xenopus oocytes, intact reticulocytes and reticulocyte lysates. The inhibition affects general protein synthesis, is transient in oocytes and not reversed by oestrogen. The drug appears to act by inhibiting polypeptide chain elongation. This action of tamoxifen is independent of oestrogen receptors and may explain its therapeutic effectiveness in oestrogen-independent tumours.