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H M Seyfert

Publications and source records attributed to H M Seyfert.

At least 19 recordsLinked to original sources
PubMed2002

STAT5 binding contributes to lactational stimulation of promoter III expressing the bovine acetyl-CoA carboxylase alpha-encoding gene in the mammary gland.

Activity of acetyl-CoA carboxylase (ACC)-alpha is rate limiting for de novo synthesis of fatty acids. The encoding gene is expressed by three different promoters. We characterized promoter III (PIII) from cow, previously only known from sheep. Quantitation of transcripts by RNAse protection assays and real time PCR revealed that PIII is primarily expressed and strongly induced ( approximately 28-fold) in the lactating mammary gland. PIII transcripts are expressed in mammary epithelial cells (MEC) as shown by in situ hybridization. A 2999 bp segment of the PIII promoter conferred prolactin and dexamethasone inducibility to a luciferase reporter gene in stably transfected mouse MEC cells. Lactogenic induction was abolished if a unique signal transducer and activator of transcription (STAT)-binding site at position -797 was inactivated by two point mutations. An oligonucleotide probe harboring this STAT-site specifically bound nuclear proteins from the lactating mammary gland. Binding was abolished by those two point mutations and super-shift analyses showed that STAT5A factors are present in this complex. Hence, prolactin, acting through STAT5, contributes to the activation of ACC expression in the milk producing cells of the lactating mammary gland. We discuss that STAT5 might be important in determining the milk composition by coordinating fatty acid and protein synthesis during lactation.

Acetyl-CoA Carboxylase↗
PubMed2001

Genomic distribution of three promoters of the bovine gene encoding acetyl-CoA carboxylase alpha and evidence that the nutritionally regulated promoter I contains a repressive element different from that in rat.

The enzyme acetyl-CoA carboxylase alpha (ACC-alpha) is rate-limiting for the synthesis of long-chain fatty acids de novo. As a first characterization of the bovine gene encoding this enzyme, we established the entire bovine ACC-alpha cDNA sequence (7041 bp) and used experiments with 5' rapid amplification of cDNA ends to determine the heterogeneous composition of 5' untranslated regions, as expressed from three different promoters (PI, PII and PIII). The individual locations of these promoters have been defined within an area comprising 35 kbp on Bos taurus chromosome 19 ('BTA19'), together with the segmentation of the first 14 exons. Primer extension analyses reveal that the nutritionally regulated PI initiates transcription from at least four sites. PI transcripts are much more abundant in adipose and mammary-gland tissues than in liver or lung. A 2.6 kb promoter fragment drives the expression of reporter genes only weakly in different model cells, irrespective of stimulation with insulin or dexamethasone. Thus bovine PI is basically repressed, like its analogue from rat. Finely graded deletions of PI map two separate elements, which have to be present together in cis to repress bovine PI. The distal component resides within a well-preserved Art2 retroposon element. Thus sequence, structure and evolutionary origin of the main repressor of PI in bovines are entirely different from its functional counterpart in rat, which had been identified as a (CA)(28) microsatellite. We show that, in different mammalian species, unrelated genome segments of different origins have been recruited to express as functionally homologous PI the ancient and otherwise highly conserved ACC-alpha-encoding gene.

Acetyl-CoA Carboxylase↗
PubMed2000

Molecular characterization of STAT5A- and STAT5B-encoding genes reveals extended intragenic sequence homogeneity in cattle and mouse and different degrees of divergent evolution of various domains.

The STAT transcription factors form a family of signal transducers and activators of transcription. We sequenced the bovine STAT5B cDNA and both STAT5-encoding genes, STAT5A and STAT5B, representing the first complete description of any STAT5-encoding gene. DNA fiber FISH hybridization revealed that the genes reside only 40 kbp apart on BTA19. Both genes are segmented into 19 exons and all but two of the homologous exons are of equal size. The genes harbor a central block of nearly identical DNA sequence (97.5% sequence identity over 3373 bp), spanning from intron 5 to intron 9. Isolation and sequencing of the homologous segments from mouse revealed the same unusually high degree of intronic sequence conservation in these segments of the murine STAT5-encoding genes. However, the respective sequences are completely divergent between the two species. A comparison of the inter- and intragenic cDNA sequence preservation at nonsynonymous sites reveals that the DNA-binding domain is under the strongest selection pressure for both intergenic and factor-specific intragenic sequence preservation. The so-called "SH3" segment of the linker domain, in contrast, shows species-specific sequence identity in all but one amino acid residues in both factors, in cattle, human, and mouse. This indicates that the same species-specific selection pressure occurs on the linker domain from both factors, STAT5A and STAT5B. Thus, the comparison of evolutionary selection pressures resting on various domains suggests that the DNA-binding domain might contribute to differential DNA binding of STAT5A and STAT5B factors, while both might interact equally well with other cellular factors through a segment of the linker domain.

Amino Acid Sequence↗
PubMed1999

Recovery of 15N-lactoferrin is higher than that of 15N-casein in the small intestine of suckling, but not adult miniature pigs.

Performance of biological functions of lactoferrin in the small intestine requires at least some resistance to degradation. Therefore, we studied prececal digestibility of lactoferrin in comparison to casein both in suckling and adult miniature pigs, applying 15N-labeled proteins. In study 1, 43 piglets (10-d-old), deprived of food for 12 h received 10 mL of sow's milk supplemented with 120 mg of 15N-labeled protein (porcine or bovine lactoferrin or bovine casein). Piglets were anesthetized 150 min later, after which the small intestine was excised, cut into three sections, and chyme was collected. In study 2, nine food-deprived boars fitted with T-canulae at the terminal ileum were given two semisynthetic experimental meals (204 g) in a cross-over design, 2 wk apart. One contained 7.5% (g/100 g) 15N-labeled bovine casein, the other 1.25% 15N-labeled bovine lactoferrin. Both were adjusted to 15% total protein with nonlabeled casein. Ileal chyme was collected from the canula over 33 h postprandially. All diets contained the indigestible marker chromic oxide. 15N-digestibility of lactoferrin, both porcine (84.4 +/- 3.2%) and bovine (82.3 +/- 4.8%), was significantly lower than casein digestibility (97.6 +/- 0.5%) in the distal small intestine of suckling piglets (P < 0.05). Based on immunoblotting after acrylamide electrophoresis, 4.5% of non- and partially digested lactoferrin was found in the last third of the small intestine of piglets. In adult miniature pigs there was no difference in 15N-digestibility of bovine lactoferrin compared to bovine casein (90.7 +/- 1.9% vs. 93.9 +/- 1.0%, P > 0.05). In suckling miniature pigs, the reduced digestibility of lactoferrin may provide the prerequisite for biological actions along the whole intestinal tract. The source of lactoferrin, porcine or bovine, made no difference in this respect.

Aging↗
PubMed1998

Cloning and sequencing of the bovine STAT5A cDNA reveals significant sequence divergence with ovine.

The transcription factors STAT5 mediates prolactin signals in mammary epithelial cells. The cDNA of bovine STAT5A was cloned, sequenced and compared to other species. The encoded protein proves to be > 95% homologous to other mammals. We show that the STAT5A mRNA of the closely related ovine species contains an extended (by 130 nt) 5'-untranslated region, being encoded by an extra-exon, and accounts, possibly, for improved translation efficiency.

Amino Acid Sequence↗
PubMed1998

Co-segregation of alleles at a microsatellite locus within the macrophage expressed lysozyme gene and levels of serum lysozyme activity in two half-sib families of Polish black and white lowland cattle.

Alleles at a microsatellite locus within the macrophage expressed lysozyme gene were shown to co-segregate with lysozyme activity in two half-sib families of Polish Black and White Lowland cattle. The bimodal distribution of lysozyme activities in both progeny groups is concordant with the occurrence of the alternative paternal alleles. The microsatellite is linked to a locus for high lysozyme activity that accounts for 70-95% of the phenotypic variation of both offspring groups considering the lysozyme activities of animals being older than 1 month.

Alleles↗
PubMed1996

Structural deviations in a bovine low expression lysozyme-encoding gene active in tissues other than stomach.

Lysozyme-encoding genes (Lys) constitute a gene-family in ruminants. While several of these genes are highly expressed in stomach (sLys), few other copies are weakly expressed in other tissues, notably in polymorphnuclear granulocytes and macrophages (mLys). Searching an understanding for these grossly different levels of expression, we isolated the bovine variant of the gene being expressed in granulocytes and characterized it by sequencing, together with its promoter. Spanning about 9 kb of genomic DNA, the gene is found to be segmented into four exons, in common with all other Lys, as known from vertebrates. Sequence homologies between all bovine sLys-variants exceeds 70% over much of the entire coding sequence and promoter region. This indicates (i) that bovine lysozymes expressed either in stomach or granulocyte originate from a common ancestral gene and (ii) also excludes the possibility that the observed weak expression of the mLys gene is due to major structural rearrangements within the promoter segment. However, primer extension analysis based on RNA isolated from kidney locates the transcription startpoint (tsp of that gene) 44 nt further upstream than observed in both, bovine stomach lysozyme RNA or any of the homologous genes in mice and man. The observed weak expression of this bovine mLys gene appears to be a consequence of both the presence of an extra ATG codon in the extended 5'-UTR, and a severe down mutation of the ancestral TATA-box which is only partially compensated for by the presence of another mutation further upstream resulting in a weak substitute promoter sequence.

Animals↗
PubMed1996

Structure of the growth hormone-encoding gene and its promoter in mice.

The isolation and nucleotide sequence determination of the 5' flanking region of the mouse growth hormone (mGH)-encoding gene (mGH) is described. The mGH gene consists of five exons and four introns, as is observed in other mammalian species. The second intron in mGH is much smaller than its rat counterpart, thus being similar in size to human, bovine and porcine GH. The transcription start point was determined to be a C residue 62 bp upstream from the start codon, ATG. Analysis of 1767 bp of the 5' flanking region, with respect to putative regulatory elements, revealed a TATA box, two binding sites for growth hormone factor (GHF1), a GC box (SP1), a thyroid-response element (TRE) and a silencer (SiL) sequence motif. As expected, the mGH promoter shows a higher degree of homology with rat, as compared to the other mammalian species like pig, cattle and human, where an overall homology exists only at the proximal promoter region.

Amino Acid Sequence↗
PubMed1994

Structure of the bovine lactoferrin-encoding gene and its promoter.

Lactoferrin (Lf), a ferric ion (Fe3+)-binding glycoprotein, is found most notably in milk, probably to mediate protection against microbial infection of the mammary gland. Based on an initial isolation and sequencing of a complete cDNA of the bovine Lf gene (bLf), the complete gene was obtained from genomic libraries on five overlapping phage lambda EMBL3 clones. A detailed restriction map and the complete exon/intron structure of the gene are presented, together with 1 kb of sequence data of the promoter upstream from the proximal exon. The coding sequence is dispersed over 17 exons spanning 34.5 kb of genomic DNA. While the exons are of similar size, as in other members of the transferrin gene family (Tf), some of the intron sizes are very different. Evolutionary conservation of both exon sizes and their contribution to the various domains of the protein molecule add to the evidence that Lf originated via an internal sequence duplication. The promoter sequence lacks some of the sequence motifs for transcriptional enhancers found in the promoters of human and mouse Lf, suggesting a potential reason for the relatively weak expression of bLf.

Animals↗
PubMed1994

Lysozyme-encoding bovine cDNAs from neutrophile granulocytes and mammary gland are derived from a different gene than stomach lysozymes.

cDNA copies of a bovine lysozyme (bLys)-encoding gene (Lys) were isolated from libraries specific for granulocytes, as well as the lactating mammary gland. Analysis of each of the longest Lys-specific cDNA inserts revealed nucleotide sequence identity over the entire overlap of 1418 bp. Incomplete at the 5' end, the combined sequence codes for 11 of the 18-amino-acid (aa) Lys leader peptide and 130 aa residues of the mature Lys. Similar to mouse and human Lys from blood cells, the encoded protein contains one aa residue more (Pro103) than any of the bLys derived from stomach. Furthermore, unlike any of the known bLys genes, our sequence reveals the copy of a bovine retroposon element in the 3' untranslated region (UTR) of the mRNA approximately at the same position where an Alu-retroposon element resides within the human copy of the gene. As a further distinction from bLys expressed in stomach, we identified a segment within the 3'UTR of the mRNA which is conserved between the bovine and human blood cell variants of the Lys, but does not have significant sequence homology to any of the bovine lysozyme genes known so far. By sequence comparisons, we present evidence that this segment has been deleted during evolutionary divergence of the stomach Lys. Hence, we describe the sequence of a heretofore unknown bLys, being expressed in granulocytes. Bearings of our observations on the understanding of Lys evolution are discussed, as well as the possibility that the product of this gene may be responsible for the functional Lys activity in bovine milk.

Amino Acid Sequence↗
PubMed1994

The bovine lactoferrin gene (LTF) maps to chromosome 22 and syntenic group U12.

Five overlapping lambda EMBL-clones, containing the complete bovine lactoferrin gene (LTF), have been used to map this gene by fluorescence in situ hybridization to bovine Chromosome (Chr) band 22q24. Primers derived from promoter and exon I sequences were applied in polymerase chain reactions (PCRs) to DNA samples of a previously characterized panel of somatic cell hybrid lines, allowing the assignment of the bovine lactoferrin locus to syntenic group U12. These results permit the assignment of syntenic group U12 to bovine Chr 22.

Animals↗
PubMed1993

Definition, distribution, and use of a conserved Bovidae retroposon element sequence motif.

Based on a data-base search, the sequences of 32 Bovidae retroposon elements have been compared. Two conserved areas are identified, and one of the corresponding sequences of the derived bovine consensus was used to design oligonucleotides as primer molecules for random DNA amplification of Bovidae DNA. Such a primer binding site should occur on average every 10,000 bp in the bovine genome, as suggested by a survey of published sequences. This estimate about the distribution of these possible primer binding sites was experimentally substantiated by mapping four of these primer binding sites within 40 kb of contiguous bovine DNA, carrying the heretofore undescribed bovine lactoferrin gene. Furthermore, these conserved, ubiquitous sequence motifs prove to be useful for mapping of bovine DNA.

Animals↗