Longitudinal bone growth occurs in the epiphyseal growth plate and is regulated by a network of paracrine and autocrine interactions. Bone morphogenetic proteins (BMPs) are a family of growth factors whose potent osteogenic properties suggest that they may play an important role within this network, but direct evidence for this is lacking. To address this question, a cDNA encoding chick BMP-7 was cloned from a chick embryo cDNA library. Sequence homology and evolutionary arguments strongly suggested that we had cloned the chicken BMP-7 homologue. Using a reverse transcription-PCR assay, BMP-7 expression was readily detected in bone, growth plate cartilage, brain and heart, and was just detectable in liver, skeletal muscle and adipose tissue. In contrast to the pattern of BMP-7 expression in the rat and mouse, no BMP-7 expression was detected in the chick kidney. In situ hybridization was used to locate the site of BMP-7 expression more precisely within the growth plate. BMP-7 expression was confined to hypertrophic chondrocytes adjacent to and at the tips of the metaphyseal vessels. No expression was detected in the reserve zone or in proliferating chondrocytes. These results point to a specific role for BMP-7 in the growth plate, possibly in osteoblast activation or as a chemotactic agent for the metaphyseal vessels.

Amino Acid Sequence↗

PubMed1992

Multiple growth factor mRNAs are expressed in chicken adipocyte precursor cells.

We have examined the expression of growth factor genes in primary cultures of chicken adipocyte precursors. RNA was extracted from proliferating and differentiated cells, reversed transcribed and amplified by PCR using gene specific primers. The identity of the PCR products was confirmed by restriction mapping. We show, for the first time, constitutive expression of TGF-beta 2, TGF-beta 3, TGF-beta 4 and bFGF genes in chicken adipocyte precursors. We also detect GH-independent, but differentiation-dependent IGF-I gene expression. The synthesis and action of these growth factors supports the hypothesis that they act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation.

Adipose Tissue↗

PubMed1992

Evolutionary grouping of the transforming growth factor-beta superfamily.

TGF beta 1 is the prototype of a superfamily of differentiation factors with at least 18 distinct members. This classification is based on amino acid homology in the C-terminus of these polypeptides. The rates of amino acid substitution for several family members were estimated from a comparison of homologous sequences derived from different species. These rates were approximately constant for any given protein, but values varied greatly between different groups. This variation is a reflection of the great functional diversity found within the TGF beta superfamily. Maximum parsimony analysis allowed us to classify members of the TGF beta superfamily into five main groups (INH alpha, MIS, TGF beta s, INH beta s and BMPs) and various subgroups. This classification predicts possible phylogenetic relationships among these proteins. In the future, it is hoped that this method of classification will be adopted by all our colleagues, as an aid in deciding whether newly discovered proteins are the product of duplicated or homologous genes. This would suggest proteins with either similar or identical functions.

Amino Acid Sequence↗

PubMed1992

Evolutionary origins of the transforming growth factor-beta gene family.

A molecular phylogeny for the transforming growth factor-beta (TGF-beta) gene family based on a comparison of nucleotide sequences is proposed. A phylogenetic tree constructed from these sequences shows that the family evolved from a common ancestral gene that came into existence at about the time of arthropod and chordate divergence. This model suggests that the present day TGF-beta gene family consists of four members: TGF-beta 1 (= TGF-beta 4), TGF-beta 2, TGF-beta 3, and TGF-beta 5. The molecular phylogeny and Southern hybridization data also suggest that the proteins for mammalian TGF-beta 1 and chicken TGF-beta 4 are the products of homologous rather than duplicated genes. If the gene duplication event that produced the ancestral gene for TGF-beta 1 occurred before the divergence of birds and mammals, then sufficient time would have elapsed to generate these quite distinct avian and mammalian TGF-beta 1 proteins. Therefore, the TGF-beta family contains four distinct proteins, TGF-beta 1, 2, 3, and 5.

Amino Acid Sequence↗

PubMed1992

Estimation of restriction maps with known site order using a generalized linear model.

A generalized linear model with Gamma errors is used to estimate the coordinates of a restriction map when the site order is known. This can be conveniently programmed in a wide range of statistical packages (e.g. Genstat 5, Minitab, SAS), and gives maximum likelihood estimates with their associated optimal properties. Regression diagnostics allow the checking of assumptions and help to identify mis-specified, influential or discordant fragment lengths. A specific diagnostic for identifying fragment lengths causing reversal of restriction site order is derived. Exact 'fragment' lengths from DNA sequencing can be conveniently included in an approximate manner by giving them a larger weight than observed restriction fragment lengths. Two examples and the Genstat 5 codes used in their analysis are presented.

Algorithms↗

PubMed1992

Cell-dependent posttranslational processing and secretion of recombinant mouse renin-2.

In the DBA/2 mouse submandibular gland (SMG), renin is predominantly the expression product of the renin gene Ren-2d. Prorenin is synthesized and rapidly converted to a constitutively secreted single-chain intermediate, which is then processed to and stored as the mature two-chain (2C) form, which is released by regulated secretion. To evaluate whether the mode of renin processing is cell dependent, renin (Ren-2d) complementary DNA was stably integrated in the genome of Chinese hamster ovary (CHO) cells and a mouse pituitary cell line (AtT-20) by transfection, and renin processing and secretion were examined. Transfected CHO cells secreted exclusively prorenin, whereas transfected AtT-20 cells secreted both prorenin and active renin. AtT-20 cells processed prorenin to the single-chain polypeptide (1C-renin) that was the main storage form and was not further processed to the 2C form of correct size, whose site of generation or function is uncertain at this time. In addition, the conversion of prorenin to 1C-renin was much slower in AtT-20 cells than in the SMG. Thus the patterns of renin biosynthesis and secretion in AtT-20 cells show major differences when compared with these processes in the native SMG, suggesting that cell-dependent characteristics, e.g., the presence of specific processing enzymes, are important factors influencing mouse renin processing.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Tissue specificity of renin promoter activity and regulation in mice.

Certain mouse strains (e.g., DBA/2) contain two renin genes (termed Ren-1 and Ren-2) and express higher renin levels in nonkidney tissues than strains with a single renin gene. The 5'-flanking regions of the Ren-1 and Ren-2 genes contain several TATA boxes preceding putative transcriptional start sites. These initiators are termed P1a, P1, P2 (from 5' to 3'), and their function (with the exception of P2) is largely unknown. In this study, we mapped the renin transcriptional start sites in renal and extrarenal tissues [adrenal, brain, testis, heart, and submandibular gland (SMG)] and examined the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on tissue specific promoter usage. Our results showed that, in the unstimulated state, P2 (the predicted initiator) is active in all DBA/2 mouse tissues. Additional transcriptional start sites were detected in the adrenal and testis (originated by P1a and P2) and the SMG (originated by P1a, P1, and P2). The administration of 8-bromoadenosine 3',5'-cyclic monophosphate led to selective stimulation of P1a in the adrenal but did not affect the selective usage of initiation sites in other organs. A locus-specific ddNTP primer extension assay was used to verify which renin gene is induced by cAMP. Results indicated that both Ren-1 and Ren-2 responded to cAMP treatment in identical fashion. Taken together, these data indicate that more than one form of renin transcript is present in several mouse tissues. There is tissue specificity in promoter usage in the unstimulated state and in response to cAMP.

Animals↗

PubMed1992

Correction: a new interpretation of a chicken transforming growth factor-beta 4 complementary DNA.

Explore the source record for details and available documents.

Amino Acid Sequence↗

PubMed1991

Molecular cloning and primary structure of the chicken transforming growth factor-beta 2 gene.

The chicken transforming growth factor-beta 2 (TGF-beta 2) gene and its flanking regions were cloned and characterized. The gene contains 7 exons and 6 introns spanning about 70 kb. Primer extension analysis identified one major and two minor starts of transcription. A comparison of the 5'-flanking regions for human and chicken TGF-beta 2 genes revealed limited sequence homology around the start of transcription, including conserved TATA-box, CRE, and AP-2 sequence motifs. A species comparison of the 5' untranslated region did not reveal any sequence homology beyond the coding region. In contrast, the 3' untranslated region was highly conserved, suggesting that this region may play an important role in the expression of the TGF-beta 2 gene.

Animals↗

PubMed1991

Comparative analysis of human and chicken transforming growth factor-beta 2 and -beta 3 promoters.

The promoters for chicken transforming growth factor-beta 2 (TGF-beta 2) and TGF-beta 3 were cloned and sequenced to study the regulation of these genes. The promoters are GC-rich and lie within CpG islands. Several putative DNA regulatory sequence motifs were identified in the 5'-flanking regions, including matches to particular recognition sequences for several nuclear factors found in other genes. A comparison of chicken and human TGF-beta 2 promoters revealed a 111 bp conserved sequence surrounding the major transcription start site. Two regions of sequence homology were detected in the 5'-flanking regions of chicken and human TGF-beta 3 genes: an 86 bp sequence surrounding the major transcription start and a 156 bp sequence in the 5'-untranslated region. No DNA sequence homology was detected between TGF-beta 1, -beta 2 or -beta 3 promoters. The conserved region near the major transcription start sites in both the TGF-beta 2 and TGF-beta 3 genes, however, does show some structural homology; both promoters contain short conserved sequences that resemble TATA box, cyclic AMP-responsive element and AP-2 sequence motifs, cis-acting elements we believe may be important for promoter activity.

Amino Acid Sequence↗

PubMed1989

The nucleotide sequence of a mouse renin-encoding gene, Ren-1d, and its upstream region.

The renin-encoding genes have been cloned from high (Ren-1d, Ren-2d)- and low (Ren-1c)-renin-producing strains of mice (DBA/2J and C57BL/10). Each of the genes is approx. 9.6 kb in length and consists of nine exons and eight introns. The entire nucleotide sequence of the Ren-1d gene has been determined and the 5'-flanking regions of the three genes, Ren-1c, Ren-1d and Ren-2d, have been compared. The significance of several potential regulatory signals found in the DNA is discussed.

Amino Acid Sequence↗

PubMed1989

Identification of negative and positive regulatory elements in the human renin gene.

Renin gene expression is tissue-specific and under complex hormonal control. To investigate which DNA elements are involved in the control of human renin gene expression, we performed transient DNA transfer experiments with renin-chloramphenicol acetyltransferase fusions. We have mapped a complex arrangement of positive and negative control sequences in the 5' flanking region of the human renin gene. One positive control element is active in either orientation and defines a renin gene enhancer. The negative element is also active in either orientation and defines a renin gene silencer. Mapping in the same region as the silencer is a cAMP-responsive element, a sequence conserved in mouse, rat, and human renin genes.

Animals↗

PubMed1989

Negative control elements and cAMP responsive sequences in the tissue-specific expression of mouse renin genes.

The 5' flanking regions of the mouse renin genes (Ren1d and Ren2d) contain putative negative control and cAMP responsive elements. Sequence analysis shows additionally that these putative control elements in the Ren2d gene are interrupted by a 160-base-pair insertion. To document the functions of these elements, we isolated these regions and fused them to the reporter gene chloramphenicol acetyltransferase (CAT), which was linked upstream to a thymidine kinase (TK) promoter (pUTKAT1). The chimeric constructs were transfected into mouse pituitary tumor AtT-20 and human choriocarcinoma JEG-3 cells. At the basal unstimulated condition, Ren1d 5' flanking sequence in the sense orientation inhibited basal CAT expression from the TK promoter of pUTKAT1, whereas the same sequence in the antisense orientation did not. The 5' flanking region of Ren2d had no inhibitory effect on basal CAT expression. These data demonstrate that the negative control element is functional in Ren1d but is nonfunctional in Ren2d, suggesting that the 160-base-pair insertion in Ren2d interferes with the function of the negative control elements. In response to 8-bromo-cAMP, both renin genes increased transcription 3-fold, suggesting a functional cis action of the cAMP responsive element in both genes. These data may be important in the understanding of the regulation of the tissue-specific expression of mouse renin genes.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1989

Identification of cis-acting sequences responsible for phorbol ester induction of human serum amyloid A gene expression via a nuclear factor kappaB-like transcription factor.

We have analyzed the 5'-flanking region of one of the genes coding for the human acute-phase protein, serum amyloid A (SAA). We found that SAA mRNA could be increased fivefold in transfected cells by treatment with phorbol 12-myristate 13-acetate (PMA). To analyze this observation further, we placed a 265-base-pair 5' SAA fragment upstream of the reporter chloramphenicol acetyltransferase (CAT) gene and transfected this construct into HeLa cells. PMA treatment of these transient transfectants resulted in increased CAT expression. Nuclear proteins from PMA-treated HeLa cells bound to this DNA fragment, and methylation interference analysis showed that the binding was specific to the sequence GGGACTTTCC (between -82 and -91), a sequence previously described by R. Sen and D. Baltimore (Cell 46:705-716, 1986) as the binding site for the nuclear factor NF kappa B. In a cotransfection competition experiment, we could abolish PMA-induced CAT activity by using cloned human immunodeficiency virus long-terminal-repeat DNA containing the NF kappa B-binding sequence. The same long-terminal-repeat DNA containing mutant NF kappa B-binding sequences (G. Nabel and D. Baltimore, Nature [London] 326:711-713, 1987) did not affect CAT expression, which suggested that binding by an NF kappa B-like factor is required for increased SAA transcription.

Base Sequence↗