Development of multidrug resistance type I Cmdr1 expression in chicken embryonic gonads

A primary role of P-glycoprotein (P-gp), encoded by the multidrug resistance type I gene, is to protect against naturally occurring xenotoxics. Recently, the preferential expression of chicken multidrug resistance type I (Cmdr1) was identified in the embryonic gonads during the early periods of development. Here we investigated the expression of Cmdr1 and P-gp in the gonads during embryogenesis, and compared to that in the ovarian follicles of domestic hens (Gallus gallus). As revealed by immunohistochemistry, P-gp was highly expressed in theca cells of mature follicles, whereas the expression was low in immature follicles. Immunohistochemical analysis showed that expression of Cmdr1-type P-gp was very low in embryonic gonads. Cmdr1 mRNA was undetectable in the gonads of 5-day embryos (E5) by RT-PCR, whereas Cmdr1 mRNA was significantly detectable in the developing gonads at E9 and E21. In the testicular tissues, germ cells were distributed along developing seminiferous cords as identified by a specific marker gene, whereas Cmdr1-type P-gp positive cells were observed evenly on testicular tissues. Collectively, it is concluded that Cmdr1 expression is initiated in the chicken ovary and testis after sexual differentiation, but expression of Cmdr1-type P-gp is very low through embryogenesis.     

Molecular Cloning of a Novel Gene ZAhi-1 and its Expression Analysis during Zebrafish Gametogenesis

Proto-oncogen Ahi-1 is closely related to a lot of human and mouse diseases. Ahi-1 mutation will lead to leukemia in mice and Joubert syndrome in human beings. We have cloned the full cDNA sequence of Ahi-1 homologous in zebrafish, and RT-PCR results of ZAhi-1 in different tissues reveal that ZAhi-1 expressed highest in the mature gonad. In situ hybridization results of zebrafish gonad show that ZAhi-1 only expressed in the early stages’ gamete cells. RT-PCR analyses of mouse Ahi-1 in different stages of spermatogenesis have been done according to the published Ahi-1 sequence, and the findings reveal that Ahi-1 is expressed in gamete of pachytene stage. It can then be safely concluded that Ahi-1 might take place in the spermatocyte from the early pachytene stage to the late pachytene stage.     

Erythropoietin gene expression in haemopoietic cell lines

Erythropoietin (Epo) gene expression was studied in a number of different haemopoietic cell lines by in situ hybridization and Northern Blot analysis using a radioisotope-labelled monkey Epo DNA probe. A positive message was expressed by a human cell line, CM-S, derived from a patient with congenital hypoplastic anemia, and by a murine erythro-leukaemic cell line, clone 707, derived from the spleen of Friend virus-infected mice. No message was detected in two megakaryoblastic cell lines, and in a monocytic cell line, derived from a patient with acute monocytic leukaemia. These data may fit with the hypothesis of expression of Epo and other growth factors by haemopoietic cells through a mechanism of so-called autocrine secretion.     

Molecular cloning, expression and characterization of the zebrafish bram1 gene, a BMP receptor-associated molecule

Summary We have identified a cDNA clone encoding BMP receptor-associated molecule 1 (BRAM1) from the zebrafish expressed sequence tag (EST) database. The 2606 bp full-length bram1 cDNA was cloned, and further confirmed by nucleotide sequencing. The zebrafish sequence encodes a protein of 195 amino acids with an evolutionarily conserved MYND domain, which displays ∼ ∼98% homology with human and mouse BRAM1, and ∼ ∼64% homology with C. elegans BRA-1 and BRA-2. The bram1 gene, composed of five exons and four introns, spans ∼ ∼14 kb on linkage group 14 of the zebrafish genome. RT-PCR and whole mount in situ hybridization analyses disclosed that zebrafish BRAM1 is a maternal factor. The protein interacts directly with zebrafish BMP Receptor type IA, as observed from GST-pull down and co-immunoprecipitation assays. Furthermore, cotransfection of zebrafish BRAM1 with the corresponding BMP receptor resulted in down-regulation of BMP-mediated signaling. Our results collectively indicate that BRAM1 plays a biological role during zebrafish development.     

The myrosinase gene family in Arabidopsis thaliana: gene organization,expression and evolution

Myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1.) is in Brassicaceae species such as Brassica napus and Sinapis alba encoded by two differentially expressed gene families, MA and MB, consisting of about 4 and 10 genes, respectively. Southern blot analysis showed that Arabidopsis thaliana contains three myrosinase genes. These genes were isolated from a genomic library and two of them, TGG1 and TGG2, were sequenced. They were found to be located in an inverted mode with their 3 ends 4.4 kb apart. Their organization was highly conserved with 12 exons and 11 short introns. Comparison of nucleotide sequences of TGG1 and TGG2 exons revealed an overall 75% similarity. In contrast, the overall nucleotide sequence similarity in introns was only 42%. In intron 1 the unusual 5 splice border GC was used. Phylogenetic analyses using both distance matrix and parsimony programs suggested that the Arabidopsis genes could not be grouped with either MA or MB genes. Consequently, these two gene families arose only after Arabidopsis had diverged from the other Brassicaceae species. In situ hybridization experiments showed that TGG1 and TGG2 expressing cells are present in leaf, sepal, petal, and gynoecium. In developing seeds, a few cells reacting with the TGG1 probe, but not with the TGG2 probe, were found indicating a partly different expression of these genes.     

Long-term organ culture of mouse mammary gland

Summary A method for maintaining mouse mammary gland in organ culture for periods of at least 30 days is described. Strips of the number four mammary glands were cultured in individual tubes while fully submerged in Medium 199 supplemented with insulin, aldosterone, ovine prolactin and bovine growth hormone. Exchange processes were aided by slowly rotating the tubes during culture. Mammary tissue from midpregnant BALB/c and virgin GR/A mice was induced to undergo lobulo-alveolar development, secrete and remain differentiated and metabolically active for the period of culture. Cells of both the ductal and alveolar epithelium continued to synthesize DNA and divide. The submerged roller-tube culture allows the use of larger pieces of tissue than can be accommodated in static culture, and the technique may prove applicable to the culture of a variety of tissues.     

Antisense inhibition of butyrylcholinesterase gene expression predicts adverse hematopoietic consequences to cholinesterase inhibitors

Summary 1. To investigate the possibility that cholinesterase inhibitors may cause adverse hematopoietic effects, we employed antisense oligodeoxynucleotides selectively inhibiting butyrylcholinesterase gene expression (AS-BCHE). Complementary sense (S) oligonucleotides served as controls.2. In primary bone marrow cell cultures grown with interleukin 3 (IL-3), AS-BCHE but not S-BCHE reduced growth of megakaryocyte colony-forming units (CFU-MK) in a dose-dependent manner at the micromolar range.3. In cultures grown with IL-3, transferrin, and erythropoietin (Epo), cell counts increased up to twofold, yet colony counts (CFU-GEMM) remained unchanged under AS-BCHE treatment.4. Electrophoretic measurements of DNA ladder as an apoptotic index revealed that the above oligonucleotide effects were not due to nonspecific induction of programmed cell death.5. Differential cell counts demonstrated increased myeloidogenesis and reduced levels of early megakaryocytes in CFU-GEMM under AS-BCHE, suggesting requirement of the BuChE protein for megakaryopoiesis.6.In vivo injection of AS-BCHE reduced BCHE mRNA levels in both young and mature megakaryocytes for as long as 20 days, as shown byin situ hybridization.7.Ex vivo growth of primary bone marrow cells revealed a twofold reduction in CFU-MK colonies grown from the AS-BCHE- but not the S-BCHE-injected mice, 15 days posttreatment.8. These findings demonstrate that deficient butyrylcholinesterase expression, and hence interference with this enzyme's activity through treatment with or exposure to cholinesterase inhibitors, may cause hematopoietic differences in treated patients.     

A novel human hydroxysteroid dehydrogenase like 1 gene (HSDL1) is highly expressed in reproductive tissuesa

We report the cloning and characterization of a novel human hydroxysteroid dehydrogenase like gene (HSDL1) located on human chromosome 16q24.2. The HSDL1 cDNA is 3407 base pair in length, encoding a 309 amino acid polypeptide related to human 17-HSD3. Northern blot reveals that the HSDL1 is highly expressed in testis and ovary. In situ hybridization indicates that the expression of HSDL1 is predominantly increased in the prostate cancer tissue compared with the normal prostate tissue, which suggests that the gene expression is important to the arising of prostate cancer.     

Development of hamster two-cell embryos in the isolated mouse oviduct in organ culture system

Hamster early two-cell embryos developed to the expanded blastocyst stage within the isolated mouse ampulla maintained in organ culture system. Mouse ampullae isolated at different times after treating the mice with human chorionic gonadotropin (hCG) (0–72 h) or pregnant mare's serum gonadotropin (PMSG) (30–32 h) were flushed with culture medium, and hamster early two-cell embryos were introduced into these ampullae. Mouse ampullae isolated at 14–32 h after hCG injection were more favorable for the development of the embryos than those isolated at 70–72 h. When mouse ampullae were isolated 30–32 h after hCG or PMSG treatment, 39% of the cultured eggs developed, some of them to the expanded blastocyst stage after additional culture for 65–70 h. These results indicate that unknown oviductal factors stimulate the development of hamster early two-cell embryos, and these factors are under the control of hCG or PMSG. In addition, these factors are common to the mouse and hamster.     

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.     

Tissue-specific expression of the rolA gene mediates morphological changes in transgenic tobacco

The spatial and temporal activity of the entire and individual promoter domains of the rolA gene of Agrobacterium rhizogenes was investigated and correlated with the distinctive features of the phenotypes of transgenic tobacco plants. The GUS assay was performed in the presence of an oxidative catalyst during the development of transgenic plants expressing chimeric genes containing the -glucuronidase coding sequence under the control of the different promoter domains. In situ hybridization was also used on transgenic plants harbouring rolA under the control of the entire or deleted promoter. This paper demonstrates for the first time that the entire rolA promoter, composed of domains, A, B and C, is silent in seeds, then activated at the onset of germination in the cotyledons and in the elengation zone of the radicle and is finally expressed throughout the vegetative and floral phases. Domains B+C, which were sufficient to induce wrinkled leaves and short internodes, were active in all the stem tissues, but only in the companion cells of the phloem strands of the leaves. Domain C, which specified a dwarf phenotype with normal leaves, was weakly expressed in the stem vascular bundles and in the leaf internal phloem. These results indicate that the vascular bundles are the primary targets for the generation of the short internode phenotype. Furthermore, the local expression of rolA in the stem vascular bundles induced a size reduction of the surrounding parenchyma cells, suggesting the existence of some diffusible factor(s) associated with the expression of the rolA gene.     

The use ofin situ hybridization histochemistry for the study of neuropeptide gene expression in the human brain

1. The application of in situ hybridization histochemistry to the study of neuropeptide gene expression in human brain postmortem tissues is reviewed. We focus on neuropeptides preferentially expressed in hypothalamus and basal ganglia. 32P-labeled oligonucleotides were used as hybridization probes. 2. Autoradiography combined with computerized image analysis was used to visualize and quantify the hybridization signal. 3. Several criteria were considered in order to ascertain the specificity of the signal, including Northern analysis, use of heterologous probes, competition assays, and thermal stability of the hybrids. 4. In control human striatum high levels of hybridization signal were observed for somatostatin, neuropeptide Y, and preproenkephalin A mRNAs. In contrast, no detectable signal was observed with the cholecystokinin, arginine-vasopressin, and oxytocin probes in this area. In the hypothalamus high levels of oxytocin and arginine-vasopressin mRNAs were visualized in several nuclei. Preproenkephalin A and somatostatin mRNAs were also observed in this region, while cholecystokinin mRNA was not detected. 5. No significant correlations were found between the density of the hybridization signal and parameters such as postmortem delay, age, and gender in the population studied. 6. Finally, alterations of mRNA levels for some of these peptides were found in Parkinson's disease and Huntington's chorea striatal tissues. 7. These results show that in situ hybridization histochemistry can be used to examine at the microscopic level neuropeptide gene expression in postmortem materials.     

Anther-specific,developmentally regulated expression of genes encoding a new class of proline-rich proteins in sunflower

We have used RNA gel blot analysis to demonstrate the anther-specific expression of three genes in sunflower. Expression of these genes was first detected shortly before flower opening, which occurs sequentially on the sunflower inflorescence, and continues during pollination. In contrast, these genes are not expressed (or only weakly expressed) in a male-sterile line in which anther development aborts. In situ hybridization experiments showed that these genes are only expressed in the single cell layer of the sunflower anther epidermis. In the case of one of these genes, which codes for an abundant mRNA, we report the peptide sequences deduced from the sequence of two similar but non identical cDNAs. These proteins contain a potential signal peptide and are characterized by the presence of a proline-rich region which reads KPSTPAPPPPPP(PP)K. Our results also suggest that several proline-rich proteins of unknown functions are specifically synthesized during the maturation of anthers in sunflower.