Comparison of the tissue-specific expression and developmental regulation of two closely linked rodent genes encoding cytosolic retinol-binding proteins

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are two highly homologous cytoplasmic proteins that bind all-trans-retinol. We have recently demonstrated that the mouse genes encoding CRBP and CRBP II are closely linked on chromosome 9 and that both human genes are located on chromosome 3 (Demmer, L.A., Birkenmeier, E.H., Sweetser, D.A., Levin, M.S., Zollman, S., Sparkes, R.S., Mohandas, T., Lusis, A.J., and Gordon, J.I. (1987) J. Biol. Chem. 262, 2458-2467). We have now used RNA blot hybridization analysis to assess the degree to which these genes are coordinately expressed in fetal, suckling, weaning, and adult rat tissues. Both genes exhibit different developmental patterns of expression in liver, intestine, lung, kidney, testes, and placenta. In the intestine, CRBP mRNA was detected during the 16th day of gestation--prior to the development of a well-differentiated absorptive epithelium--and remained essentially unchanged throughout the peri- and postpartum periods. By contrast, the pattern of intestinal CRBP II mRNA accumulation closely parallels the times of first appearance, and subsequent proliferation, of the intestinal absorptive columnar epithelium, supporting the hypothesis that CRBP II is involved in the intestinal uptake or intracellular trafficking of this hydrophobic vitamin. In the fetal liver, both genes were expressed by gestational day 16. Whereas the concentration of hepatic CRBP mRNA increased markedly during the suckling and early weaning periods, CRBP II mRNA levels fell abruptly immediately after birth. These peripartum changes were not paralleled by remarkable alterations in the steady state levels of hepatic retinol. Marked changes in the expression of CRBP in the liver and of CRBP II in the intestine were also documented in pregnant and lactating female rats. These differences in CRBP/CRBP II gene expression strongly suggest that their proteins serve different physiological functions. The peripartum liver may provide a useful model for dissecting the relative roles played by these homologous proteins in retinoid metabolism as well as the factors which modulate activation and repression their genes.     

Cloning and tissue expression of chicken heart fatty acid-binding protein and intestine fatty acid-binding protein genes

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.     

Cloning and Tissue Expression of Chicken Heart Fatty Acid-Binding Protein and Intestine Fatty Acid-Binding Protein Genes

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.     

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.     

Tissue specific expression and developmental regulation of two genes coding for rat fatty acid binding proteins

We have examined the tissue distribution and developmental regulation of two low molecular weight cytosolic fatty acid binding proteins. Based on their initial site of isolation, they have been referred to as liver and intestinal fatty acid binding proteins (FABP). Cloned cDNAs were used to probe blots of RNAs extracted from a wide variety of adult rat tissues as well as small intestine and liver RNA obtained from fetal, suckling, and weaning animals. The highest concentrations of "liver" FABP mRNA were found in small intestine and liver. "Intestinal" FABP mRNA is most abundant in small bowel RNA while only trace amounts were encountered in liver. Both mRNAs were detectable in stomach, colon, pancreas, spleen, lung, heart, testes, adrenal, and brain RNA at 1-8% the concentrations observed in small intestine. Accumulation of both mRNAs in the small intestinal epithelium increases during development. The mRNAs are first detectable between the 19th and 21st day of gestation. They undergo a coordinated 3-4-fold increase in concentration within the first 24 h after birth. Thereafter, gut levels of intestinal FABP mRNA remain constant during the suckling period while liver FABP mRNA increases an additional 2-fold. Liver FABP mRNA levels are also induced in hepatocytes during the first postnatal day but subsequently do not change during the suckling and weaning phase, despite marked alterations in hepatic fatty acid metabolism. These observations support the concept that the major role of these proteins is to facilitate the entry of lipids into cells and/or their subsequent intracellular transport and compartmentalization. The data also raise questions about the identity of extragastrointestinal FABPs.     

Abundance patterns of lily pollen cDNAs: characterization of three pollen-preferential cDNA clones

Twenty-five clones were randomly selected from a mature pollen cDNA library of Easter lily (Lilium longiflorum Thunb.) in order to study the abundance of pollen-expressed mRNAs and the functional roles of the proteins encoded by these mRNAs. Plaque hybridization experiments were conducted to estimate indirectly the expression level of the mRNAs. Based on the hybridization frequency in the mature pollen library, the cDNA clones were divided into three abundance groups. Eight clones belonged to a high abundance class in which each cDNA clone was present in the mature lily pollen library at a frequency between 0.3 and 3%. Six of these clones were not found in cDNA libraries made from carpel, leaf, or root, suggesting that they are preferentially expressed in pollen. Fourteen clones belonged to a medium abundance class and were present in the mature pollen library at a frequency between 0.01 and 0.08%. The remaining three clones, which were present at a frequency below 0.01%, were grouped as a low abundance class. Almost all of the cDNA clones which belong to either the medium or low abundance class were also detected in the leaf library. Northern blot hybridization with three of the highly abundant cDNA clones confirmed their preferential expression in anther. In situ hybridization experiment with one of the clones showed the pollen-specific expression of the clone in mature anther. DNA sequence analysis revealed that the clone LMP131 encodes a peptide which is highly homologous to the tomato pollen-preferential gene, LAT59, which encodes a putative pectate lyase. The clone LMP134 encodes a peptide that shows an extensive similarity to a variety of thioredoxins. The third clone LMP132 encodes a 182-residue protein that has no significant homology to known sequences.     

Molecular cloning of mRNAs expressed specifically during spherulation of Physarum polycephalum

A cDNA library was constructed using the poly(A)+ RNA extracted from spherulating Physarum polycephalum microplasmodia. This library (740 clones) was screened by differential hybridization with 32P-labeled poly(A)+ RNA from growing plasmodia and developing spherules. The results showed that at least 30% of the clones corresponded to mRNAs expressed specifically in spherulating plasmodia. The 35 spherulation-specific cDNA clones giving the strongest hybridization signals were analysed. From this group, four different sequences complementary to very abundant mRNAs were identified. They each accounted for 1.5% of 4.5% of all the clones in the library and probably represented the most abundant spherulation-specific mRNAs. In addition, four less abundant mRNAs were identified from stage-specific clones giving weaker hybridization signals. These sequences represented individually between 0.3% and 0.7% of the clones in the library. Northern blots showed that these eight different sequences were absent from plasmodia and were most abundant 24-36 h after the induction of spherulation. Similar results were also obtained when spherulation was induced by the addition of a sublethal concentration of ferrous iron ions to the growth medium. Hybridization of the spherule-specific clones to Southern blots of genomic DNA suggested the presence of one copy for each gene.     

1,25-Dihydroxyvitamin D3-stimulated mRNAs in rat small intestine

The technique of differential hybridization has been employed to study gene expression associated with vitamin D action on the mammalian intestine. A cDNA library consisting of 10(6) independent recombinants was constructed from poly(A)+ RNA extracted from vitamin D-deficient rats given 1,25-dihydroxyvitamin D3. A survey of 20,000 clones resulted in identification of four distinct cDNAs whose corresponding mRNAs are significantly increased 12 h after an intrajugular dose of 1,25-dihydroxyvitamin D3 given to vitamin D-deficient rats. DNA sequence analysis identified these mRNAs as mitochondrial ATP synthetase, vitamin D-dependent calcium binding protein, cytochrome oxidase subunit I, and cytochrome oxidase subunit III. The time course of response of three of these mRNAs was similar, with maximum values at 12 h after dosing, while that of cytochrome oxidase subunit I showed two peaks at 6 and 18 h following a single dose of 1,25-dihydroxyvitamin D3. The levels of all four mRNAs were elevated in rats supplied with vitamin D when hypocalcemia was produced by dietary calcium restriction.     

Identification and characterization of rat intestinal keratins. Molecular cloning of cDNAs encoding cytokeratins 8, 19, and a new 49-kDa type I cytokeratin (cytokeratin 21) expressed by differentiated intestinal epithelial cells.

In the previous paper (Quaroni, A., Calnek, D., Quaroni, E., and Chandler, J.S. (1991) J. Biol. Chem. 266, 11923-11931) we describe the use of a panel of "antikeratin" monoclonal antibodies to study cytokeratin distribution in rat intestinal epithelium. In the present paper we describe the use of three antikeratin monoclonal antibodies to identify and recovery cDNA clones expressing immunologically specific fusion proteins from a rat intestinal cDNA library. DNA sequence analysis identified each cDNA encoded epitope including the carboxyl-terminal portions of cytokeratins 8 and 19 (as cataloged by Moll, R., Franke, W.W., and Schiller, D.L. (1982) Cell 31, 11-24) recognized by antibodies RK4 and RK7, respectively. In addition, antibody RK5 was used to recover a cDNA clone (pRK5) encoding a portion of a 48-kDa keratin-related protein with unique tissue and cellular distribution, designated cytokeratin 21. Translation of cDNA-selected mRNAs yielded individual proteins which could be resolved and identified by their specific immunoreactivities. The pRK5 cDNA was used to recover a larger (approximately 1.3 kilobase pairs) cDNA clone (KB2) from an independent cDNA library for DNA sequence analysis and for the recovery of additional overlapping cDNA clones. The resulting cDNA sequence (1519 base pairs) contains the complete coding region of cytokeratin 21 (49,387 daltons). The predicted amino acid sequence of cytokeratin 21 confirms its identity as a novel type I cytokeratin expressed predominantly in the intestinal epithelium.     

Identification of putative parasitism genes expressed in the esophageal gland cells of the soybean cyst nematode Heterodera glycines

Cloning parasitism genes encoding secretory proteins expressed in the esophageal gland cells is the key to understanding the molecular basis of nematode parasitism of plants. Suppression subtractive hybridization (SSH) with the microaspirated contents from Heterodera glycines esophageal gland cells and intestinal region was used to isolate genes expressed preferentially in the gland cells of parasitic stages. Twenty-three unique cDNA sequences from a SSH cDNA library were identified and hybridized to the genomic DNA of H. glycines in Southern blots. Full-length cDNAs of 21 clones were obtained by screening a gland-cell long-distance polymerase chain reaction cDNA library. Deduced proteins of 10 clones were preceded by a signal peptide for secretion, and PSORT II computer analysis predicted eight proteins as extracellular, one as nuclear, and one as plasmalemma localized. In situ hybridization showed that four of the predicted extracellular clones were expressed specifically in the dorsal gland cell, one in the subventral gland cells, and three in the intestine in H. glycines. The predicted nuclear clone and the plasmalemma-localized clone were expressed in the subventral gland cells and the dorsal gland cell, respectively. SSH is an efficient method for cloning putative parasitism genes encoding esophageal gland cell secretory proteins that may have a role in H. glycines parasitism of soybean.     

Novel sperm-binding proteins of epididymal origin contain four fibronectin type II-modules

Novel fibronectin type II (Fn2)-module proteins were cloned from human and canine epididymal cDNA libraries. cDNA sequences predicted a highly conserved protein family, related but not homologous to ungulate seminal plasma proteins (approximately 50% sequence identity), and the first known examples of proteins with four tandemly arranged Fn2-domains. By Northern blot and in situ hybridization analyses the encoding mRNAs were shown to be abundant products of the epididymal duct epithelium, but not detectable in other tissues. Homologous mRNAs were identified in the epididymides of various mammals, representing members of this novel protein family of epididymal origin. Within the Fn2-module-encoding stretches, species homologues displayed >85% sequence identity, but showed high variability at their predicted N-termini. An antipeptide antiserum in Western blot analyses detected 30-35 kDa immunoreactive protein bands in epididymal tissue, cauda epididymidal fluid, and sperm membrane protein preparations. The tandem arrangement of increasing numbers of Fn2-modules might functionally correspond to the tendency to form oligomers that has been described for lipid-binding proteins.