Heparin-binding growth factor type 1 (HBGF-1; sometimes termed acidic fibroblast growth factor) is potentially an important factor in liver regeneration. HBGF-1 alone (half-maximal effect at 60 pM) stimulated hepatocyte DNA synthesis and bound to a high-affinity receptor (Kd = 62 pM; 5000 per cell). Epidermal growth factor (EGF) neutralized or masked the mitogenic effect of HBGF-1 concurrent with appearance of low-affinity HBGF-1 binding sites. HBGF-1 reduced the inhibitory effect of transforming growth factor type .3 (TGF-.B) on the EGF stimulus. Nanomolar levels of HBGF-1 decreased the EGF stimulus. An increase in hepatic HBGF-1 gene expression after partial hepatectomy precedes increases in expression of the EGF homolog, TGF-a, and nonparenchymal-celi-derived TGF-fi in the regenerating liver.Expression of HBGF-1 mRNA occurs in both hepatocytes and nonparenchymal cells and persists for 7 days in liver tissue after partial hepatectomy. HBGF-1 acting through a high-affinity receptor is a candidate for the early autocrine stimulus that drives hepatocyte DNA synthesis prior to or concurrent with the EGF/TGF-et stimulus. It may allow hepatocyte proliferation to proceed in the presence of low levels of TGF-,B. An EGF/TGF-ae-dependent change in HBGF-1 receptor phenotype and increasing levels of nonparenchymal-cell-derived HBGF-1 and TGF-,B may serve to limit hepatocyte proliferation.Adult hepatocytes rarely divide except in response to injury or xenobiotics. After partial hepatectomy, DNA synthesis in hepatocytes begins about 14 hr after the operation and peaks at 24 hr (1, 2). The liver remnant doubles after 36 hr. By 1 week 90% of the liver mass is restored, and hepatocytes return to quiescence. Liver cell DNA synthesis, cell division, and return to quiescence is thought to be controlled by growth-stimulatory and growth-inhibitory hormones of autocrine and paracrine origin (3)(4)(5) Hepatocyte Cultures. Liver of adult male F344 rats (200-250 g) were perfused through the portal vein with Hepesbuffered saline (pH 7.0) containing 5 mM EGTA and then were perfused for 10-15 min with the same solution containing 0.05% (wt/vol) collagenase, 1 mM calcium chloride, and soybean trypsin inhibitor (10 ,ug/ml). The perfused liver was excised and dispersed in Hepes-buffered saline. The cell suspension was filtered through gauze, and hepatocytes were collected by centrifugation (four times) at 50 x g for 1 min. Hepatocytes were plated at 1 x 105 cells in 35-mm plastic Petri dishes coated with type I/III collagen containing 3 ml of nutrient medium MCDB 107 (14) and 2% (vol/vol) fetal bovine serum. After 2 hr, the medium was removed and replaced with serum-free medium containing bovine serum albumin (1 mg/ml), oleic acid (4 ,ug/ml), dithiothreitol (50 ,.uM), ethanolamine (5 ,ug/ml), and insulin (1 jig/ml). HBGF-

The abundant class of poly(A+)RNA [poly(A+)RNA ] from rat seminal vesicle was used to synthesize ds-cDNA1 . The ds-ckAA

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The rat seminal vesicle secretion IV (SVS IV) gene was isolated from a A Charon 4A library. The SVS IV gene transcription unit was found to be on one 3.3-kilobase (kb) EcoRI fragment. Restriction mapping and DNA sequence analysis demonstrated that the entire length of the SVS IV transcription unit is 1,930 base pairs (bp) and contains two introns. The 3.3-kb EcoRI fragment contains 144 bp of 5'-flanking region. At -113 bp from the presumed transcription initiation site an interesting structure with perfect dyad symmetry is noted. In another A clone, a 3.5-kb EcoRI fragment was isolated that contains the SVS IV gene and was shown to be identical to the 3.3-kb EcoRI fragment except for 180 bp of DNA in the second intron. The extra DNA consists of several (8-10) 20-bp tandem repeats flanked on each side by seven or eight copies of this same 20-bp repeat. Fisher X Sprague-Dawley hybrid rats, which contain both the EcoRI 3.5-kb form and the 3.3-kb form of the SVS IV gene, were crossed with each other. Analysis of the F1 generation demonstrated that the presence or absence of the 180-bp intronic insertion in the SVS IV gene defines an allelic difference. This report also presents the DNA sequence of the transcription unit and flanking regions of the SVS IV gene.The rat seminal vesicle, under the influence of testosterone, produces a group of small basic polypeptides referred to as seminal vesicle secretion IV and V (SVS IV and V) (1, 2). We have isolated an llS poly(A)+ mRNA fraction that codes for precursors to SVS IV and V (3). The complementary DNA (cDNA) to the mRNA was cloned in Escherichia coli by using plasmid pBR322. Clones were identified and cDNAs were sequenced that contain DNA information for SVS IV and V mRNA (4, 5). It was shown, by using SVS IV cDNA as probe, that the mRNA for SVS IV is rapidly induced after testosterone administration to rats castrated for several weeks (4, 6).One clone, pSV2, has a cDNA insert complementary to SVS IV mRNA and 540 base pairs (bp) long (4). The cDNA insert from pSV2 was used as a probe to screen a rat gene library prepared in phage A Charon 4A. Several positive phage signals were identified and the rat DNA constituting the genomic DNA sequences for the SVS IV gene was then characterized by restriction mapping and DNA sequence analysis. To our surprise, the SVS IV gene exists in two forms, which differ by the number of 20-bp tandem repeats in an intron. This intron size variability in the SVS IV gene was shown to be an allelic variant by the appropriate genetic crosses. A major repetitive element was also identified just 3' to the end of the transcription unit. 32P-Labeled DNA probes were prepared by nick-translation (4). To identify genomic clones of the SVS IV gene, approximately 3 million plaques of the A Charon 4A library of rat genomic DNA were screened by the Benton-Davis procedure (7), with an amplification step as recommended by S. Woo (8) Electrophoresis of DNA on agarose gels, Southern transfers, and hybridizations were performed as described (10, 11)...

A 1.2 kb HBGF-1 cDNA was isolated from a rat prostate transplantable Dunning tumor 3327 AT-3 cDNA library using the human HBGF-1 cDNA (1) as a probe. The nucleotide sequence shown in fig. 1 was determined by sequencing overlapping cDNA clones, combined with new primers, by the chain termination method. gcccagggtg ggaggaaggg cggtaatctc taggaagcag aaggcaggtt tgcagaggac gctggatagg agatgaggtg 80 ggggaaacca aggcctgggc ttcccgcagc tgcagcaagg ttttggtgct taccacagca gcaggaatgc attgagggat 160 gtgacagtgg aacgcaggtg gcagtgatac tcactgctgc tgaaggagcc caaagaacca gcatctgacc tgtcttgaac 240 gcaccatcga cagttgctgc tgagtcATGG CCGAAGGGGA GAGCACAACC TTTGCAGCCC TGACCGAGAG GTTCAATCTG 320 CCTCTAGGGA ACTACAAAAA ACCCAAACTG CTCTACTGCA GCAACGGGGG CCACTTCTTG AGGATTCTTC CCGATGGCAC 400 CCTGGATGGG ACCAGGGACA GGAGCGACCA GCACATTCAG CTGCAGCTCA GTGCGGAAAG CGCGGGCGAA GTGTATATAA 480 AGGGTACAGA GACTGGCCAG TACTTGGCCA TGGACACCGA AGGGCTTTTA TACGGCTCGC AGACACCAAA TGAAGAATGC 560 CTATTCCTGG AAAGGCTAGA AGAAAACCAT TATAACACTT ACACATCCAA GAAGCACGCG GAGAAGAACT GGTTTGTGGG 640 CCTCAAGAAG AACGGGAGTT GTAAGCGCGG TCCTCGGACT CACTACGGCC AGAAAGCCAT CTTGTTTCTC CCCCTCCCGG 720 TATCTTCTGA CTAAggagtc tgttctgggt gctccctatt tttggttgac cctaaaatgt tcccttgacc attggctgcg 800 ctaaccctca gcccacagag cctgaatttg taagcagtgc ttctasatag ccogttcact tttccgctga gcccttcccc 880 accaccccag cacagtttgg aacacgggga ccaaattact tctaggggac aactggctgg ccagtctagg tctgggtttg 960 gatatccaga tgcttcaggg tacctgctac aagttggacc tcgtgataca aagaggggag ccatgtgtag catctggagg 1040 ggtctaagaa gtgggttctt ttagtagctg catgctcttt ccccttctga gtaatccctg gttttgagta cccccaggca 1120 cttagcatga ctacccttcc ccctaccagc acctcataaa tatcagtaaa gaaactgata gtaaaaaaaa (a)25 1216 Fig. 1 Nucleotide sequence of rat HBGF-1.Rat HBGF-1 consists of 156 amino acids, the same as human and bovine HBGF-1 (1,2,3). There is 95% homology between rat and human HBGF-1 and 90O homology between rat and bovine. The amino acid comparision is shown in fig. 2. Fig. 2 Amino acid sequence of human, rat and bovine HBGF-1. Amino acid differences are indicated by an asterisk.