Characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase expressed in the adrenal gland and gonads

The guinea pig adrenal gland, analogous to the human, possesses the capacity to synthesize C(19) steroids. In order to further understand the control of guinea pig adrenal steroidogenesis we undertook the characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD) expressed in the adrenal gland. A cDNA clone encoding guinea pig 3beta-HSD isolated from a guinea pig adrenal library is predicted to encode a protein of 373 amino acid residues and 41,475Da. Ribonuclease protection assay suggests that this cDNA corresponds to the predominant, if not the sole, mRNA species detectable in total RNA from the guinea pig adrenal gland, ovary and testis. The guinea pig 3beta-HSD shows a similar affinity for both pregnenolone and dehydroepiandrosterone, and in addition, a 17beta-HSD type II-like activity was also observed. A phylogenetical analysis of the 3beta-HSD gene family demonstrates that the guinea pig is in a parallel branch to the myomorpha group supporting the hypothesis that the guinea pig lineage has branched off after the divergence among primates, artiodactyls and rodents, suggesting the paraphyly of the order rodentia.     

A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase

The subcellular location of several nonribosomal nucleolar proteins was examined at various stages of mitosis in synchronized mammalian cell lines including HeLa, 3T3, COS-7 and HIV-1 Rev-expressing CMT3 cells. Nucleolar proteins B23, fibrillarin, nucleolin and p52 as well as U3 snoRNA were located partially in the peripheral regions of chromosomes from prometaphase to early telophase. However, these proteins were also found in large cytoplasmic particles, 1–2 μm in diameter, termed nucleolus-derived foci (NDF). The NDF reached maximum numbers (as many as 100 per cell) during mid- to late anaphase, after which their number declined to a few or none during late telophase. The decline in the number of NDF approximately coincided with the appearance of prenucleolar bodies and reforming nucleoli. The HIV-1 Rev protein and a mutant Rev protein defective in its nuclear export signal were also found in the NDF. The mutant Rev protein precisely followed the pattern of localization of the above nucleolar proteins, whereas the wild-type Rev did not enter nuclei until G1 phase. The nucleolar shuttling phosphoprotein Nopp 140 did not follow the above pattern of localization during mitosis: it dispersed in the cytoplasm from prometaphase through early telophase and was not found in the NDF. Although the NDF and mitotic coiled bodies disappeared from the cytoplasm at approximately the same time during mitosis, protein B23 was not found in mitotic coiled bodies, nor was p80 coilin present in the NDF. These results suggest that a class of proteins involved in preribosomal RNA processing associate with chromosome periphery and with NDF as part of a system to conserve and deliver preexisting components to reforming nucleoli during mitosis. Edited by: S. A. Gerbi     

Mycosporine glutamine and related mycosporines in the fungus Pyronema omphalodes

The structure of mycosporine glutamine, a new compound, has been established and its presence demonstrated in two fungi Pyronema omphalodes and Glomerella cingulata. Mycosporine glutamic acid has been isolated from Helvella leucomelaneae. Co-occurrence of normycosporine glutamine, mycosporine glutamine and glucosylmycosporine glutaminol has been demonstrated in the fungus P. omphalodes. A biosynthetic pathway is proposed. Mycosporines have been compared by HPLC.     

Rabbit whey acidic protein gene upstream region controls high-level expression of bovine growth hormone in the mammary gland of transgenic mice

Transgenic mice were produced which secreted high levels of bGH into milk. The 6.3-kb upstream region of the rabbit whey acidic protein (rWAP) gene was linked to the structural part of the bovine growth hormone (bGH) gene, and the chimeric gene was introduced into mouse oocytes. bGH was detected by radioimmunoassay in the milk of all resulting transgenic mice. bGH concentrations in milk varied from line to line, from 1.0–16 mg/ml. This expression was not correlated to the number of transgene copies. In all lines studied, the mammary gland was the major organ expressing bGH mRNA during lactation. bGH mRNA concentrations were barely detectable in the mammary gland of cyclic females; they increased during pregnancy. These results show that the upstream region of the rWAP gene harbors powerful regulatory elements which target high levels of bGH transgene expression to the mammary gland of lactating transgenic mice. © 1995 wiley-Liss, Inc.     

Patterns of integration of exogenous DNA sequences transfected into mammalian cells of primate and rodent origin

We studied the cotransfer and cointegration of several genes transfected into four cell lines of primate origin. Mouse thymidine-kinase-negative LM cells, which had been extensively studied previously, were used as a reference. We found that in monkey kidney Vero cells, on average between 3.5 and 6.0 kb of plasmid sequences was integrated per clone, while in the murine LM cell Une, 9–186 kb of exogenous DNA was integrated per clone. Transformed Vero clones which had integrated more than 6 kb of DNA did not integrate larger DNA fragments in a second transformation assay than had the parental Vero cells. We found that the efficiency of gene cointegration is similar in Vero, HeLa and GM4312A cells, the latter being deficient in the repair of UV-induced damage. The human hepatocarcinoma Hep G2 cells integrated on the average 2 kb more exogenous DNA than the three other primate cell Unes, which resulted in a 4–5 times higher efficiency of gene cointegration. Plasmid penetration and persistence in a free state between 24 h and two weeks after transfection was similar in Vero and LM cells. No major post-integration DNA rearrangement could be demonstrated after the isolation of Vero clones. These observations correlate the low efficiency of gene cointegration in some primate cell lines with a genomic recombination step or with rearrangements taking place during early cell divisions following integration     

Immunogold distribution of actin during sperimiogenesis in the normal rabbit and after experimental cryptorchidism

Immunogold procedures for actin detection were used in combination with experimental cryptorchidism in the rabbit as a modei to shed more light on the function of subacrosomal actin during spermiogenesis. In the normal testis, actin was localized in the perinuclcar substance (PNS) from round spermatid onward but it was not detected in late spermatids. Actin labeling in each type of spermatid was essentially unmodified after 24 hr of cryptorchidism. However, among relevant immediate and delayed effects, discontinuous acrosomes overlying a continuous PNS with normal actin labeling were noted. Nuclear invaginations were seen in combination with subacrosomal dilatations: at this site actin labeling was found only in the PNS closely apposed to the nuclear envelope. In subacrosomal areas lacking PNS, actin labeling also was lacking. These results suggest that the subacrosomal actin (F-actin) is a component of the PNS that is tightly bound to the nuclear envelope rather than the overlying inner acrosomal membrane. Therefore, a function for the subacrosomal actin either in anchoring the acrosome to the nucleus or in capping the inner acrosomal membrane appears unlikely. The data rather suggest a capping function for the nuclear membrane during spermiogenesis.     

The brain ofRhyacichthys aspro (Rhyacichthyidae,Gobioidei)

Rhyacichthys aspro has one of the highest encephalization indices of the Gobioidei, at the level of the amphibiousPeriophthalmus (Gobiidae, Oxudercinae). This high encephalization can be explained by its adaptation to the turbulent waters of mountain torrents. The brain morphology is typical of a perciform fish and similar to that of a gobioid except in the form and size of the cerebellum. The quantitative analysis of the brain structures shows a large size of the olfactory centers, a small size of the visual centers (compared to those of other Gobioidei) and a very large size of the cerebellar centers (more than twice the size in other Gobioidei). The brain organization shows thatRhyacichthys aspro, although some of its brain structures are typically gobioid (tegmentum, medulla oblongata), is not a generalized gobioid, because of the high degree of its biological adaptation and the correlated large size of its cerebellum.