Phylogenetic information in inter-SINE and inter-SSR fingerprints of the artiodactyla and evolution of the bov-tA SINE.

Various interspersed repeated sequences and elements (IRSs) can be utilized to generate PCR-based multilocus fingerprint profiles by amplifying the interelement segments, using primers matching the elements themselves. We assessed the utility of inter-IRS fingerprinting in phylogenetic comparisons among six artiodactyl species using several primers derived from two abundant genomic components: the Bov-tA short interspersed nuclear elements (SINEs) and simple sequence repeats or microsatellites (SSRs). Character- and distance-based analyses of the fingerprint data produced trees conforming to the established phylogenetic relationships of species. The strength of phylogenetic signal from different primers varied; combining data from different experiments resulted in robust trees. Within the Cervidae, the hierarchical relationship [(Odocoileus, Rangifer) Alces] was strongly supported. Both methods appear useful tools for systematic studies at time scales <30 Myr. To elucidate the material basis of inter-SINE fingerprints, we obtained the first sequences of the 'bovid' Bov-tA element also from two cervids (reindeer and white-tailed deer) and analysed their relationship to a number of paralogous bovid elements. The differences among sequences, both intra- and interspecific, were relatively high (mean 18.5%); the sequences showed no clear clustering with the species from which they had been isolated. Most individual elements probably date back to the cervid-bovid ancestor >25 Myr ago, which is in line with the observed fingerprint distributions.

Variable number of pig MHC class I genes in different serologically defined haplotypes identified by a 3'-untranslated region probe.

To estimate the number of porcine class I major histocompatibility genes, a short class I cDNA probe from the 3'-untranslated region was developed to be used in restriction fragment length polymorphism analysis. Six clones isolated from a pig spleen cDNA library were sequenced from their 3'-untranslated region. Three different transcripts were identified, one probably derived from the class I PD7 locus and two showing highest homology to the PD1 and the PD14 genes, respectively. Class I typing was performed both by restriction fragment length polymorphism and serology. Segregation of class I haplotypes was followed in one three-generation family (European Wild Boar x Large White: Swedish Yorkshire) and in six two-generation families (Duroc, Yorkshire and Chester White), for a total of 266 pigs. Twenty different class I haplotypes were identified either with restriction fragment length polymorphism and/or serological typing. Furthermore, previously unpublished serological haplotypes H62, H67 and H68 were identified. Two to seven polymorphic and three monomorphic fragments were detected in different restriction fragment length polymorphism haplotypes indicating that the number of class I genes in the investigated haplotypes varies.

Restriction fragment length polymorphisms at the heat shock protein HSP70 gene(s) in pigs.

Pigs from a population consisting of eight US breeds or strains and three Chinese breeds were examined by restriction fragment length polymorphism (RFLP) analysis of the heat shock protein HSP70 gene(s). Limited polymorphisms with PstI and PvuII restriction enzymes were observed, but there were no polymorphisms with BamHI and BglI.

An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences.

The construction of a mammalian cell expression vector using human cytomegalovirus immediate early gene enhancer to initiate transcription of inserted coding sequences is described. The vector also carries Epstein-Barr virus EBNA-1 nuclear antigen gene, ori-P sequences and hygromycin B resistance gene hph from E. coli. The expression capacity of this construct was tested by inserting the chloramphenicol acetyltransferase (CAT) gene into the vector. The EBV-CAT construct was transfected into various cell lines and high levels of CAT activity were obtained in human and monkey cells. In these cells, the vector DNA also replicates as an extrachromosomal element having 1 to 20 copies per cell. In most cases, the vector copy number and the expression level of inserted gene was in positive correlation in different cell clones.