Vascular endothelial growth factor A improves quality of matured porcine oocytes and developing parthenotes.

Vascular endothelial growth factor is a multipotent angiogenic factor implicated in cell survival and proliferation. The objective was to determine effects of exogenous recombinant human VEGFA (or VEGFA165) in culture media on porcine oocyte maturation and parthenote development. Adding 5 ng/mL VEGFA to the culture medium improved the maturation rate of denuded oocytes (P < 0.05), although 5, 50, or 500 ng/mL did not significantly affect nuclear maturation of oocytes. Parthenotes from oocytes cultured either in in vitro maturation or in vitro culture medium supplemented with 5 or 50 ng/mL VEGFA had an improved blastocyst rate and increased total numbers of cells (P < 0.05). Moreover, those treated with 5 ng/mL of VEGFA had a higher hatched blastocyst rate (average of 121 cells per blastocyst). All VEGFA-treated oocytes had reduced apoptotic indices (P < 0.05), except for those with a higher dose (500 ng/mL) of VEGFA which had more apoptotic cells (P < 0.05). Adding 5 ng/mL VEGFA to oocytes during the last 22 h of in vitro maturation improved (P < 0.05) blastocyst rates and total numbers of cells, with reduced apoptosis indices similar to that of long-term (44 h) culture. Furthermore, Axitinib (VEGFR inhibitor) reversed the effects of VEGFA on parthenote development (P < 0.05). Follicular fluids from medium (2-6 mm) to large (>6 mm) follicles contained 5.3 and 7.0 ng/mL vascular endothelial growth factor protein, respectively, higher (P < 0.05) than concentrations in small (<2 mm) follicles (0.4 ng/mL). Also, VEGFA and its receptor (VEGFR-2) were detected (immunohistochemistry) in growing follicles and developing blastocysts. In addition, VEGFA inhibited caspase-3 activation in matured oocytes (P < 0.05). In conclusion, this is apparently the first report that VEGFA has proliferative and cytoprotective roles in maturing porcine oocytes and parthenotes. Furthermore, an optimal VEGFA concentration promoted porcine oocyte maturation and subsequent development.

Overexpression of the alpha2,6-sialyltransferase, ST6Gal I, in a low metastatic variant of a murine lymphoblastoid cell line is associated with appearance of a unique ST6Gal I mRNA.

Multiple mRNA isoforms are generated from Siat1, the gene encoding ST6Gal I (beta-galactoside alpha2,6-sialyltransferase, SiaT-1, ST6N, alpha2,6ST). These isoforms, transcriptionally initiated from a number of physically distinct promoter regions, differ only in the 5'-most untranslated region and share an identical ST6Gal I coding region. W16 cells, a spontaneous mutant from MDAY-D2, the highly metastatic murine lymphoid tumor cell line, is considerably less metastatic and exhibits significantly slower tumor growth characteristics [R. Takano, E. Muchmore, and J. W. Dennis (1994) Glycobiology 4, 665-674]. Takano et al. further reported that ST6Gal I mRNA in W16 is elevated 40-fold compared to the parental cells. Here, by means of 5'-RACE analysis, we demonstrate a heretofore undocumented ST6Gal I mRNA form expressed in W16 cells. This ST6Gal I mRNA contains a novel 5'-most untranslated region with 96% sequence similarity to the retroviral-like transposable element, intracisternal particle A (IAP). This observation suggests the notion that elevated ST6Gal I expression in W16 cells is the result of DNA rearrangement in the Siat1 locus. Atypical transcriptional activation of Siat1 is the result of this IAP transposition.

Transcription of the beta-galactoside alpha2,6-sialyltransferase gene (SIAT1) in B-lymphocytes: cell type-specific expression correlates with presence of the divergent 5'-untranslated sequence.

A single gene, SIAT1, encodes ST6Gal I, the sialyltransferase that mediates transfer of alpha2,6-linked sialic acids to Galbeta1, 4GlcNAc termini of N-linked glycoproteins. In vivo, multiple SIAT1 mRNA forms, differing only in the 5'-untranslated region, are expressed in a tissue-specific manner. This mRNA heterogeneity has been attributed, at least in part, to transcription from a number of physically distinct promoter regions. In mature B-lymphocytes, SIAT1 transcription initiates at P2, a regulatory region known to function only in B-lineage cells. Bacterial chloramphenicol acetyltransferase (CAT) under the control of the P2 region encompassing 415 bp 5'- and 125 bp 3' of the transcriptional initiation site is efficiently expressed in Louckes, a mature B-lymphoblastoid cell line. In contrast, CAT expression in Reh, a T-null/B-null precursor line, and in HepG2, a hepatoma line, are 14-fold and >25-fold less than in Louckes, respectively. The data is consistent with the presence of cis -acting regulatory elements residing both 5' and 3' of the P2 transcriptional initiation site. At least 370 bp of 5'-flanking sequence, coinciding with the inclusion of AP2 and NF-kappaB sites, is necessary for high level expression in Louckes. Exon sequences 3' of the transcription start site are also important for expression. A segment from(+)32 to(+)125 (position(+)1 is transcription start site) is capable of exerting promoter-like activity in Louckes, but not in Reh or HepG2. CAT expression by P2 is negligible in Reh cells. However, enhanced CAT activity is not accompanied by elevated mRNA levels. This observation is consistent with the relief of translational restraints imposed by the(+)32 to(+)125 region. Together, the data demonstrate that efficient and cell-specific transcription regulation in mature B lymphocytes is contained in a 495 bp P2 segment that is comprised of 370 bp of 5'-flanking region and 125 bp of transcribed region of Exon X.

Beta1,4-galactosyltransferase and lactose biosynthesis: recruitment of a housekeeping gene from the nonmammalian vertebrate gene pool for a mammary gland specific function.

Beta1,4-galactosyltransferase (beta4GalT-I) is a constitutively expressed trans-Golgi enzyme, widely distributed in vertebrates, which synthesizes the beta4-N-acetyllactosamine structure commonly found in glycoconjugates. In mammals beta4GalT-I has been recruited for a second biosynthetic function, the production of lactose; this function takes place exclusively in the lactating mammary gland. In preparation for lactose biosynthesis, beta4GalT-I enzyme levels are increased significantly. We show that mammals have evolved a two-step mechanism to achieve this increase. In step one there is a switch to the use of a second transcriptional start site, regulated by a stronger, mammary gland-restricted promoter. The transcript produced is distinguished from its housekeeping counterpart by the absence of approximately 180 nt of 5'-untranslated sequence. In step two, this truncated transcript is translated more efficiently, relative to the major transcript expressed in all other somatic tissues.

Transcription of the beta-galactoside alpha 2,6-sialyltransferase gene in B lymphocytes is directed by a separate and distinct promoter.

A single human gene, SIAT1, encodes the beta-galactoside alpha 2,6-sialyltransferase from which multiple mRNA isoforms are generated. In rat, expression of the hepatic mRNA isoform (Form 1) has been defined with respect to the transcriptional initiation site and promoter region. We show here that a similar hepatic SIAT1 mRNA isoform exists in human. Another human mRNA isoform, a mature B-cell-specific mRNA isoform (Form 2), was previously reported. Here, we used 5'-RACE and S1 nuclease protection analysis to define the 5'-untranslated region of Form 2 human SIAT1 mRNA. We demonstrate conclusively that Form 2 mRNA is initiated from a point completely distinct from that of Form 1 mRNA. A number of cis-acting regulatory elements residing immediately 5'of the Form 2 initiation site includes AP-1, AP-2, NF-kappa B, NF-IL6, C/EBP, and CREB. A TATAA box is also present 29 bp 5' of the transcriptional initiation site. CAT reporter gene expression from serially-truncated segments of the 5'-flanking region of the Form 2 initiation site indicates that the segment between -784 and +125 was sufficient to promote high level CAT expression in Louckes, a mature B-cell line. The 5'-flanking region to the human Form 1 initiation site is competent in expression of CAT upon transfection of the fusion construct into HepG2, a human hepatoma cell line. Cellular specificity of expression is apparently retained. Louckes cells expressed CAT efficiently from Form 2 promoter but only marginally from the Form 1 promoter. In contrast, CAT expression from Form 1 promoter is more efficient than from the Form 2 promoter in HepG2 cells.

Nucleotide sequence of the genomic RNA of bamboo mosaic potexvirus.

The complete nucleotide sequence of the genomic RNA of bamboo mosaic virus (BaMV) was determined by sequencing a set of overlapping cDNA clones and by direct sequencing of the viral RNA. The RNA genome of BaMV is 6366 nucleotides long [excluding 3'poly(A) tail] and contains six open reading frames (ORFs 1 to 6) coding for polypeptides with M(r) values of 155K, 28K, 13K, 6K, 25K and 14K, respectively. The genome organization and sizes of the encoded proteins are very similar to those of other potexviruses which have been sequenced except that ORF 6 lies completely within ORF 1. The first five putative proteins of the BaMV genome show identities ranging between 44 to 59%, 26 to 49%, 30 to 53%, 15 to 35% and 20 to 30%, respectively, to the corresponding ORFs of other members of the potexvirus group. However the putative product ORF 6 shows no significant similarity to those of other potexvirus ORF products.