Porcine beta1,4-galactosyltransferase-I sequence and expression.

Beta1,4-galactosyltransferase-I (B4GALT1), one of seven beta1,4-galactosyltransferases, is an enzyme commonly found in the trans-Golgi complex that adds galactose to oligosaccharides. In the three mammals studied to date, the B4GALT1 gene directs production of B4GALT1 protein using either of two transcription start sites. The product of the smaller transcript serves the traditional biosynthetic role in the Golgi. This form also complexes with alpha-lactalbumin, a mammary-specific protein, to form lactose synthase. In addition to a biosynthetic role, the protein translated from the longer transcript appears on the plasma membranes of some cells where it serves as a signalling receptor in cell-matrix interactions such as sperm-egg binding. The objective of this study was to sequence the protein-coding region of porcine B4GALT1 and examine the sequence for relationships to the bovine, human, murine and chicken B4GALT1 genes. The sequence for the 1203 base pair protein-coding region of porcine B4GALT1 was obtained. Analysis of the deduced protein sequences revealed that the transmembrane region displayed the highest identity between the four mammals. The catalytic domain was 84-88% identical between the porcine sequence and those of the bovine, human and mouse. The porcine protein had the lowest overall homology to the chicken amino acid sequence, 58% identity. Conservation of both transcription start sites in the porcine gene supports the existence of two isoforms. When compared to the other mammalian B4GALT1 genes, the porcine coding sequence contained a single threonine codon inserted into the region encoding the cytoplasmic domain. Two putative phosphorylation sites in the mouse cytoplasmic domain were conserved in the porcine sequence. Northern blots revealed a widely expressed 4.4 kb transcript that was more abundant in the mammary gland during lactation. These results are important for studies of the function of this unusual and important glycosyltransferase during glycoprotein biosynthesis, lactation and fertilization.

Placental defects in alpha7 integrin null mice.

The alpha7beta1 integrin is a heterodimeric transmembrane receptor that links laminin in the extracellular matrix to the cell cytoskeleton. Loss of the alpha7 integrin chain results in partial embryonic lethality. We have previously shown that alpha7 integrin null embryos exhibit vascular smooth muscle cell defects that result in cerebral vascular hemorrhaging. Since the placenta is highly vascularized, we hypothesized that placental vascular defects in alpha7 integrin null embryos may contribute to the partial embryonic lethality. Placentae from embryonic day (ED) 9.5 and 13.5 alpha7 integrin knockout embryos showed structural defects including infiltration of the spongiotrophoblast layer into the placental labyrinth, a reduction in the placental labyrinth and loss of distinct placental layers. Embryos and placentae that lacked the alpha7 integrin weighed less compared to wild-type controls. Blood vessels within the placental labyrinth of alpha7 integrin null embryos exhibited fewer differentiated vascular smooth muscle cells compared to wild-type. Loss of the alpha7 integrin resulted in altered extracellular matrix deposition and reduced expression of alpha5 integrin. Together our results confirm a role for the alpha7beta1 integrin in placental vascular development and demonstrate for the first time that loss of the alpha7 integrin results in placental defects.

An enhanced linkage map of the sheep genome comprising more than 1000 loci.

A medium-density linkage map of the ovine genome has been developed. Marker data for 550 new loci were generated and merged with the previous sheep linkage map. The new map comprises 1093 markers representing 1062 unique loci (941 anonymous loci, 121 genes) and spans 3500 cM (sex-averaged) for the autosomes and 132 cM (female) on the X chromosome. There is an average spacing of 3.4 cM between autosomal loci and 8.3 cM between highly polymorphic [polymorphic information content (PIC) > or = 0.7] autosomal loci. The largest gap between markers is 32.5 cM, and the number of gaps of > 20 cM between loci, or regions where loci are missing from chromosome ends, has been reduced from 40 in the previous map to 6. Five hundred and seventy-three of the loci can be ordered on a framework map with odds of > 1000 : 1. The sheep linkage map contains strong links to both the cattle and goat maps. Five hundred and seventy-two of the loci positioned on the sheep linkage map have also been mapped by linkage analysis in cattle, and 209 of the loci mapped on the sheep linkage map have also been placed on the goat linkage map. Inspection of ruminant linkage maps indicates that the genomic coverage by the current sheep linkage map is comparable to that of the available cattle maps. The sheep map provides a valuable resource to the international sheep, cattle, and goat gene mapping community.

Cell surface beta-1,4-galactosyltransferase-I activates G protein-dependent exocytotic signaling.

ZP3 is a protein in the mammalian egg coat (zona pellucida) that binds sperm and stimulates acrosomal exocytosis, enabling sperm to penetrate the zona pellucida. The nature of the ZP3 receptor/s on sperm is a matter of considerable debate, but most evidence suggests that ZP3 binds to beta-1,4-galactosyltransferase-I (GalTase) on the sperm surface. It has been suggested that ZP3 induces the acrosome reaction by crosslinking GalTase, activating a heterotrimeric G protein. In this regard, acrosomal exocytosis is sensitive to pertussis toxin and the GalTase cytoplasmic domain can precipitate G(i) from sperm lysates. Sperm from mice that overexpress GalTase bind more soluble ZP3 and show accelerated G protein activation, whereas sperm from mice with a targeted deletion in GalTase have markedly less ability to bind soluble ZP3, undergo the ZP3-induced acrosome reaction, and penetrate the zona pellucida. We have examined the ability of GalTase to function as a ZP3 receptor and to activate heterotrimeric G proteins using Xenopus laevis oocytes as a heterologous expression system. Oocytes that express GalTase bound ZP3 but did not bind other zona pellucida glycoproteins. After oocyte maturation, ZP3 or GalTase antibodies were able to trigger cortical granule exocytosis and activation of GalTase-expressing eggs. Pertussis toxin inhibited GalTase-induced egg activation. Consistent with G protein activation, both ZP3 and anti-GalTase antibodies increased GTP-gamma[(35)S] binding as well as GTPase activity in membranes from eggs expressing GalTase. Finally, mutagenesis of a putative G protein activation motif within the GalTase cytoplasmic domain eliminated G protein activation in response to ZP3 or anti-GalTase antibodies. These results demonstrate directly that GalTase functions as a ZP3 receptor and following aggregation, is capable of activating pertussis toxin-sensitive G proteins leading to exocytosis.

Gamete adhesion molecules.

Despite the importance of fertilization for animal production, species preservation and controlling reproduction, the molecular basis underlying fertilization is not well understood. More progress has been made in mice than in other mammals, but targeted deletion of specific genes in the mouse has often yielded unexpected results. The pig is also a useful animal to study, as large numbers of pig gametes can be acquired easily. However, it appears that the pig zona pellucida proteins that bind to spermatozoa may not be homologues of ZP3, the mouse zona pellucida protein that spermatozoa bind to. Therefore, a zona pellucida receptor on spermatozoa that is important for mouse fertilization may be redundant, along with other receptors, in pig fertilization. In this review, the important steps of fertilization in pigs are discussed and the binding of pig gametes is compared with that of mouse gametes. In addition, the molecules that may be important for gamete adhesion are considered. New technical advances and creative ideas offer the opportunity to make important advances in this crucial area.

Zona pellucida protein binding ability of porcine sperm during epididymal maturation and the acrosome reaction.

In many mammals, the first interaction between gametes during fertilization occurs when sperm contact the zona pellucida surrounding the egg. Although porcine sperm first contact the zona pellucida via their plasma membrane, the regions of the sperm surface that display zona receptors have not been determined. We have used the Alexa 488 fluorophore conjugated to solubilized porcine zona pellucida proteins to observe zona receptors on live boar sperm. Zona proteins bound live, acrosome-intact sperm on the anterior portion of the sperm head, concentrated in a thin band over the acrosomal ridge. When sperm membranes were permeabilized by fixation or acrosome reactions induced by the ionophore A23187, zona binding was extended to a broad area covering the entire acrosomal region. Zona binding proteins were present in the acrosomes of sperm from all regions of the epididymis. In contrast, zona binding sites were found on the plasma membrane of most sperm from the corpus and cauda epididymis, but on only 6% of caput epididymal sperm. In conclusion, acrosome-intact boar sperm exhibit concentrated zona protein binding over the acrosomal ridge and acquire this binding in the corpus region of the epididymis, correlating with the developmental stage at which sperm gain the ability to fertilize oocytes.

New gene assignments using a complete, characterized sheep-hamster somatic cell hybrid panel.

The generation and characterization of new sheep-hamster cell hybrids is reported from the fusion of sheep white blood cells with six different hamster auxotrophs. Selection from these and previously generated cell hybrids has led to the production of a panel of 30 hybrids covering the complete sheep genome of 28 chromosomes. Over half of the cell hybrids in this panel contain single sheep chromosomes. By complementation, the following new assignments have been made using the panel: phosphoribosyl N-formylglycinamide amidotransferase (PRFGA) to sheep chromosome (chr) 11; adenylosuccinate synthetase (ADSS) to sheep chr 12; adenylosuccinate lyase (ADSL) to sheep chr 3q; 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS) to sheep chr 16; dihydrofolate reductase (DHFR) to sheep chr 5; and adenine phosphoribosyltransferase (APRT) to sheep chr 14. The gene phosphoribosylaminoinidazole-carboxamide formyltransferase/Inosinicase (PRACFT) has now been regionally assigned to chr 2q. By isozyme analysis, phosphogluconate dehydrogenase (PGD) was assigned to sheep chr 12, anchoring the sheep syntenic group U1 to this chromosome, and mannose phosphate isomerase (MPI) was assigned to sheep chr 18. Furthermore, the chromosomal assignment of 110 microsatellites was confirmed using this cell panel.

Localization and genomic organization of sheep antimicrobial peptide genes.

Antimicrobial peptides are an abundant and diverse component of animal innate immunity. Within mammalian species, defensins and cathelicidins are the two principal antimicrobial peptide families. We identified and sequenced ten new sheep genes which encode potential antimicrobial peptides including two beta-defensins and eight cathelicidins. We mapped the two-exon beta-defensin genes to sheep chromosome 26 and the four-exon cathelicidin genes to sheep chromosome 19 using sheep-hamster somatic cell hybrids in conjunction with flow-sorted sheep chromosomes. These assignments confirm homology between sheep, cattle, mouse, and human antimicrobial peptide gene families. Contig construction for the sheep cathelicidin gene family demonstrates that three genes, OaDodeA, OaDodeB, and OaMAP-34, are present head-to-tail in a 14.5 kb region, and that four proline/arginine-rich genes, OaBac5, OaBac7.5, OaBac11, and OaBac6, are arranged head-to-tail in a region covering 30.5 kb. This richly diverse family of sheep cathelicidin peptides is encoded in a gene array which may reflect the mechanism of its evolution.

The Drosophila developmental gene fat facets has a human homologue in Xp11.4 which escapes X-inactivation and has related sequences on Yq11.2.

EST 221 derived from human adult testis detects homology to the Drosophila fat facets gene (fat) and has related sequences on both the X and Y chromosomes mapping to Xp11.4 and Yq11.2 respectively. These two loci have been termed DFFRX and DFFRY for Drosophila fat facets related X and Y. The major transcript detected by EST 221 is-8 kb in size and is expressed widely in a range of 16 human adult tissues. RT-PCR analysis of 13 different human embryonic tissues with primers specific for the X and Y sequences demonstrates that both loci are expressed in developing tissues and quantitative RT-PCR of lymphoblastoid cell lines carrying different numbers of X chromosomes reveals that the X-linked gene escapes X-inactivation. The amino acid sequence (2547 residues) of the complete open reading frame of the X gene has 44% identity and 88% similarity to the Drosophila sequence and contains the conserved Cys and His domains characteristic of deubiquitinating enzymes, suggesting its biochemical function may be the hydrolysis of ubiquitin from protein-ubiquitin conjugates. The requirement of faf for normal oocyte development in Drosophila combined with the map location and escape from X-inactivation of DFFRX raises the possibility that the human homologue plays a role in the defects of oocyte proliferation and subsequent gonadal degeneration found in Turner syndrome.

Sheep CENPB and CENPC genes show a high level of sequence similarity and conserved synteny with their human homologs.

Sheep CENPB and CENPC clones were isolated from a lung cDNA library. The DNA and predicted amino acid sequences of these clones were compared with their human and mouse homologs and shown to contain a high degree of sequence similarity. Sheep chromosomal assignments were made using a sheep x hamster somatic cell hybrid mini-panel. CENPB was assigned to sheep chromosome 13 and CENPC to chromosome 6. The previously reported assignments of CENPB and CENPC to human chromosomes 20 and 4, respectively, suggest conserved synteny between sheep chromosome 13 and human chromosome 20 and support conserved synteny between sheep chromosome 6 and human chromosome 4.

cDNA selection from 10 Mb of chromosome 21 DNA: efficiency in transcriptional mapping and reflections of genome organization.

The technique of cDNA hybridization selection has been applied individually to 16 YAC clones mapping to various regions of the long arm of human chromosome 21. YACs represented approximately 10 Mb of non-overlapping DNA, and cDNA sources included fetal brain, whole fetus, and adult testes, thymus, liver and spleen. Sequencing, Northern analysis, RT-PCR and cDNA library screenings have been used to identify and partially characterize a non-redundant set of novel genes. A preliminary analysis strategy of the selected cDNAs has proven rapid and effective for isolation of the more highly represented genes and is suitable for survey transcriptional mapping efforts. By scaling up to screen > 1000 cDNA fragments per 100 kb of YAC DNA, more rare transcripts are identified and lead to comprehensive gene maps. Strong regional variations in transcriptional activity were observed, with gene densities ranging from < 1/2000 kb to > 1/15 kb. This effort has produced a large number of new genes of potential relevance to Down Syndrome.