Molecular cloning and temporal expression during pregnancy of the messenger ribonucleic acid encoding uteroferrin, a progesterone-induced uterine secretory protein

A lambda gt11 expression library containing cDNA inserts prepared from porcine endometrial mRNA was immunologically screened by using an antiserum developed against porcine uteroferrin (Uf), a glycoprotein that has been strongly implicated in transplacental iron transport in the pregnant pig. Antibody reactive clones (lambda 4a3, 13.1, and 2.2) were isolated after screening 1.5 x 10(5) recombinant phages. Clones 4a3 and 13.1 expressed Uf antigenic determinants in beta-galactosidase fusion proteins and specifically selected antibody which reacted with Uf in immunoblots prepared from uterine cytosolic extracts. In addition, all three cDNA clones collectively contained DNA sequences that encoded an 85-amino acid peptide which corresponded to a region within the carboxyterminal portion of the Uf protein. Northern blot hybridization of these cDNAs to RNAs extracted from whole uterine tissue of pregnant pigs revealed a single uterine poly(A)+ RNA of approximately 1.7 kilobases in length, which was not found in liver and mammary tissue RNAs. The concentration of the Uf mRNA changed in a temporal fashion during pregnancy in a manner that was distinct from that of the progesterone receptor mRNAs. Highest levels of Uf mRNA were found at mid and late pregnancy (days 45-110) and were about 50-fold greater than at day 30 of pregnancy. By contrast, RIA analysis of the uterine tissue extracts showed that maximum amounts of Uf were present at day 60 and then declined sharply. Thus the pattern of Uf mRNA present in the uterus did not parallel the amount of Uf polypeptide that could be recovered from the tissue. The tissue specific and temporal regulation of Uf gene expression emphasizes that the protein plays an important role in uterine activity and/or fetal development.     

Expression of differentiated functions in the developing porcine small intestine

Heterologous cDNA clones were used as hybridization probes to define the temporal expression of intestinal functions during fetal and postnatal development in the pig. Northern hybridization analysis revealed the presence of the mRNAs for the cellular retinol binding protein CRBP II, for the digestive enzyme aminopeptidase N, and for the microvillar proteins villin and ezrin in the small intestine of both weaned and 40-day fetal pigs. The presence of these mRNAs suggests that at the end of the first third of gestation the pig fetal intestine is already exhibiting some characteristics of a differentiated epithelium. The mRNAs for the two fatty acid-binding proteins I-FABP and L-FAPB, both involved in the metabolism of long chain fatty acids, were detected only in the intestinal mRNA extracted from weaned animals, while that for the cellular retinol-binding protein CRBP I was expressed only in the fetal tissue. The temporal limits of expression of intestinal genes in the pig epithelium seem therefore more easily defined than in other experimental animals with shorter times of fetal development. To isolate pig genes expressed at different developmental stages during intestinal epithelial cell differentiation, a cDNA library was constructed from poly(A) + RNA extracted from mature pig intestine. This library was employed in the isolation of clones encoding CRBP II and L-FABP. The nucleotide sequence of the two pig cDNA clones was determined, and the sequences of the deduced proteins compared with their homologues from other species. The results of this analysis showed that the two pig clones share a high level of homology with human and rat homologues both at the DNA and at the protein level.     

Complementary DNA derived structure of the amino-terminal domain of human apolipoprotein B and size of its messenger RNA transcript

In this paper the sequence of a 5.2-kilobase (kb) cDNA covering the amino-terminal domain of human apolipoprotein B-100 (apoB-100) is reported. The cDNA-derived protein sequence provides the primary structure of 1748 amino acids. This segment of apoB-100 is more hydrophilic than hydrophobic and contains short stretches of predicted helical and beta structures that are interrupted by beta turns. Blotting analysis of RNA isolated from fetal human and adult monkey tissues and various human cell lines showed synthesis of apoB mRNA by liver and intestine and by cells of hepatic (HepG2) and intestinal (Caco-2) origin. The isolation and characterization of overlapping cDNA clones, which provide a nearly full-length copy of human apoB-100, are also reported. From the length of these clones the size of the cytoplasmic apoB mRNA is estimated to be 14.0 kb and codes for a protein of approximately 512,000 daltons.     

Porcine insulin-like growth factor-I (pIGF-I): complementary deoxyribonucleic acid cloning and uterine expression of messenger ribonucleic acid encoding evolutionarily conserved IGF-I peptides

In order to facilitate studies of insulin-like growth factor-I (IGF-I) expression during the pregnancy-associated development of uterus and mammary gland in the pig model, we have isolated several cDNA clones corresponding to porcine IGF-I (pIGF-I) mRNA. Sequence analysis of two cDNA fragments (sigf. 2 and sigf. 3) revealed an open reading frame encoding in order a putative 25 amino acid (aa) hydrophobic leader peptide, the mature (processed) 70 aa pIGF-I peptide and a 35 aa carboxy-terminal extension (E) peptide. The deduced aa sequence of the pIGF-I peptide is identical to human and bovine IGF-I but differs from that of rat and mouse at three and four residues, respectively. The sequences of the amino- and carboxy-terminal IGF extension peptides are also highly conserved among these species. Northern analysis using sigf. 3 as a probe revealed multiple IGF-I mRNAs (including species of 8000, 2300, and 1200 nucleotides in length) in uteri of pregnant pigs. Highest levels of the uterine IGF-I mRNAs were found at early pregnancy, when increased levels of immunoreactive tissue IGF-I were also observed. Mammary levels of IGF-I mRNAs and protein were considerably lower than that observed for uterus at the same time period. Thus, uterine production of IGF-I appears to be especially significant during early pregnancy in the pig when uterine growth, elevated IGF-I in uterine fluids, and rapid embryonic development are observed.     

Regulation of synthesis of uterine secretory proteins: evidence for differential induction of porcine uteroferrin and antileukoproteinase gene expression

Mechanisms regulating the expression of two pregnancy-associated proteins of the porcine uterus, namely the iron-transport protein uteroferrin (UF) and the lysosomal serine proteinase-inhibitor antileukoproteinase (ALP), were investigated by comparing the effects of estrogen (E), progesterone (P4), and conceptuses on the steady-state levels of their mRNAs. For UF, the expression of mRNA with production and secretion of the corresponding protein was also investigated. Progesterone increased the levels of endometrial UF mRNA and of secreted UF, but did not affect the levels of ALP mRNA in ovariectomized gilts that had received P4 treatment for 8 days. Estrogen inhibited the accumulation of endometrial UF mRNA, increased UF secretion in these gilts, but had no effect on levels of ALP mRNA. Administration of E to gilts on Day 11 of the cycle slightly diminished UF mRNA levels at 1 h post-E; had no effect at 6, 12, and 24 h post-E; and increased levels of secreted UF in uterine luminal fluids 24 h post-E. The presence of conceptuses increased levels of endometrial ALP mRNA and decreased UF protein in uterine luminal fluids, but did not affect levels of endometrial UF mRNA. Myometrium, endometrium, and placenta from Day 75 and Day 105 pregnant gilts were also evaluated for ALP and UF mRNA expression to determine regional expression of these steroid-regulated genes. Myometrium and endometrium expressed comparable levels of UF and ALP mRNAs within Days 75 or 105, but placenta did not express detectable levels of mRNA for either protein. Within the myometrium, UF protein is immunolocalized mostly to the inner circular and to a lesser extent to the outer longitudinal layer of smooth muscle. These results indicate that E, P4, and presence of conceptuses differentially affect endometrial expression of ALP and UF mRNAs and secretion of UF.     

The isolation of cDNA clones for human apolipoprotein E and the detection of apoE RNA in hepatic and extra-hepatic tissues.

We have isolated cDNA clones coding for apolipoprotein E (apoE) from a cDNA library prepared from adult human liver mRNA. Mixtures of 128 different oligonucleotides, 17 residues long were synthesised to be complementary to regions of the mRNA corresponding to amino acids 1-6 and 151-156. Five independent apoE clones were selected by direct screening of 5000 recombinants with the two oligonucleotide mixtures. Two overlapping clones contain the 3'-untranslated sequence, the entire coding sequence and an additional 30 bases 5' to the amino terminus of the mature protein. The DNA sequence has been determined spanning the known sites of amino acid substitutions which account for the observed protein polymorphism of apoE. Using the clones as probes in Northern blot analysis of total human liver and kidney RNAs and leucocyte poly(A)+ RNA we have detected a single species of mRNA in liver and kidney of 1.2 kb and two larger species in leucocyte RNA. The level of expression of the mRNA in kidney is approximately 10% of that in liver while the level of apoE RNA sequences in the leucocytes is less than 1% of that in the liver.     

The human pyruvate dehydrogenase complex. Isolation of cDNA clones for the E1 alpha subunit, sequence analysis, and characterization of the mRNA

cDNA clones corresponding to the entire length of mRNA for the alpha subunit of human pyruvate dehydrogenase (EC 1.2.4.1), the E1 component of the pyruvate dehydrogenase complex, have been isolated from liver cDNA libraries. Two classes of cDNA clones were obtained and these correspond to two forms of pyruvate dehydrogenase E1 alpha mRNA. Both mRNA species have been demonstrated in a variety of human tissues and cultured fibroblasts. The cDNA sequence has been determined and, from it, the protein sequence of the human E1 alpha subunit was deduced. The protein is synthesized with a typical mitochondrial import leader sequence and the peptide bond at which this sequence is cleaved after transport into the mitochondrion has been determined by direct amino acid sequencing of the mature E1 alpha subunit. The human pyruvate dehydrogenase E1 alpha subunit contains identical phosphorylation sites to those found in the corresponding porcine protein. Preliminary studies of pyruvate dehydrogenase E1 alpha mRNA in cultured fibroblasts from patients with severe pyruvate dehydrogenase deficiency have revealed considerable heterogeneity as would be expected from protein studies.     

Discovery of new human beta-defensins using a genomics-based approach

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.     

Nucleotide and protein sequences of a proteinase inhibitor from the vitelline envelope of dace (Tribolodon hakonensis) eggs

Our experimental purpose is to probe the structure(s) of the chorionic proteinase inhibitor and its cDNA sequence(s) and to develop the application of safe medicines for protection of human and other animal bodies from pathogenic microbe attacks. In this study, chorionic proteinase inhibitor protein was isolated, sequenced and used to base the design of PCR primers, which were then used to amplify DNA using RT-PCR. A cDNA clone of the protein which inhibited the activities of serine proteinases and thermolysin was obtained on the basis of mRNA extracted from ovarian tissue of dace, Tribolodon hakonensis, and the deduced amino acid sequence was determined. Chorionic proteinase inhibitor (TribSPI) peptides of about 9.0 kDa (TribSPI) and 14 kDa (TribSPI-S) were purified from vitelline envelope extracts by thermolysin-immobilized affinity-chromatography. The cloned TribSPI cDNA was 1806 bp in length, and the open reading flame (ORF) was 915 bp encoding a protein of 305 amino acid residues. The inhibitor protein had a molecular mass of 33,550 daltons and was composed of five similar domains. Each domain contained eight cysteine residues, and it's deduced amino acid sequence was only 33 approximately 34% identical to those of human and porcine antileukoproteinases (hALP and pALP, respectively). A possible binding-site for serine proteinases, Arg-Ile, was contained in three domains.     

cDNA cloning and sequence analysis of the glucose transporter from porcine blood-brain barrier

A porcine brain microvessel-derived cDNA library enriched in blood-brain-barrier-specific sequences was constructed by a subtractive cloning procedure. Two cDNA clones from this library were found to encode a glucose transport protein. These clones were used to isolate a nearly full length cDNA from a representative brain microvessel library. Analysis of the amino-acid sequence deduced from the cDNA sequence revealed 97% identity with the human HepG2 glucose carrier. Amino-acid substitutions appear to be clustered in certain regions of the polypeptide. The nucleotide sequences of the 3'-noncoding regions close to the putative polyadenylation sites are highly conserved in glucose transporter mRNAs of different species. The expression of this mRNA has been investigated in various tissues and shown to be decreased in primary cultures of brain microvascular endothelial cells.     

Cell cycle dependent expression of Plk1 in synchronized porcine fetal fibroblasts

Enzymes of the Polo-like kinase (Plk) family are active in the pathways controlling mitosis in several species. We have cloned cDNA fragments of the porcine homologues of Plk1, Plk2, and Plk3 employing fetal fibroblasts as source. All three partial cDNAs showed high sequence homology with their mouse and human counterparts and contained the Polo box, a domain characteristic for all Polo kinases. The expression levels of Plk1 mRNA at various points of the cell cycle in synchronized porcine fetal fibroblasts were analyzed by both RT-PCR and the ribonuclease protection assay. Plk1 mRNA was barely detectable in G0 and G1, increased during S phase and peaked after the G2/M transition. A monoclonal antibody was generated against an in vitro expressed porcine Plk1-protein fragment and used to detect changes in Plk1 expression at the protein level. Plk1 protein was first detected by immunoblotting at the beginning of S phase and was highest after the G2/M transition. In summary, the Plk1 expression pattern in the pig is similar to that reported for other species. The absence of Plk1 mRNA and protein appears to be a good marker for G0/G1 and thus for the selection of donor cells for nuclear transfer based somatic cloning.     

Molecular cloning and characterization of human atrial and ventricular myosin alkali light chain cDNA clones

We have isolated essentially full-length cDNA clones for atrial (ALC1) and ventricular (VLC1) myosin alkali light chains from a human fetal heart cDNA library. Comparison of overall nucleotide sequences of ALC1 and VLC1 cDNA clones has revealed that, while these two inserts show significant DNA sequence homology (78.4%) with respect to their coding regions, the 5'- and 3'-untranslated regions are highly divergent. Our statistical analysis suggests that human ALC1 and VLC1 diverged approximately 300 million years ago, during the time of separation of birds and mammals. RNA blot analysis shows that ALC1 mRNA is expressed in fetal ventricular and fetal skeletal muscles as well as fetal and adult atrial muscles and VLC1 mRNA is expressed in adult slow skeletal muscle as well as fetal and adult ventricular muscles. Southern blot analysis indicates that each protein is encoded by a single gene. Finally, we show that VLC1 mRNA is induced in pressure-overloaded human atrium.     

Human apolipoprotein B: identification of cDNA clones and characterization of mRNA.

Apolipoprotein B (apoB) is a major protein component of low density and very low density lipoproteins. Because of its large size and heterogeneity, molecular studies of apoB have been difficult, and its structure and regulation remain poorly understood. We now report the identification of human apoB cDNA clones by antibody screening of hepatoma libraries in the expression vector lambda gt11. Both oligo(dT) primed and random primed libraries were constructed and screened with polyclonal antibodies to intact apoB, as well as with antibodies raised against a synthetic peptide based on the limited amino acid sequence available for apoB. The identity of the clones was unambiguously established by comparisons of the cloned cDNA sequences with apoB amino acid sequences. The clones hybridize to an exceptionally large 20 kb mRNA that is present in liver and intestine but not other tissues examined, consistent with the distribution expected from protein biosynthetic studies. The properties of the mRNA have implications for the biogenesis of the multiple apoB molecular weight forms secreted by liver and intestine.     

The isolation and characterisation of cDNA and genomic clones for human lecithin: cholesterol acyltransferase

The protein sequencing of tryptic peptides from purified human lecithin: cholesterol acyltransferase (LCAT) identified sufficient amino-acid sequence to construct a corresponding mixed oligonucleotide probe. This was used to screen an adult human cDNA liver library, from which incomplete cDNA clones were isolated. The DNA sequence of these clones allows the prediction of the entire amino-acid sequence of the mature LCAT enzyme. The mature protein consists of 416 amino acids and contains several marked stretches of hydrophobic residues and four potential glycosylation sites. The cDNA probe detects LCAT mRNA sequences approx. 1500 bases long in human liver, but not intestine, RNA. The cDNA probe was used to isolate LCAT genomic recombinants from a human genomic library. Southern blotting data, and restriction site mapping, suggest that there is a single human LCAT structural gene between 4.3 and 5.5 kb in size.