A full length N‐ras gene has been cloned from both the human fibrosarcoma cell line HT1080 and from normal human DNA. N‐ras isolated from HT1080 will efficiently induce morphological transformation of NIH/3T3 cells in a transfection assay, whereas N‐ras isolated from normal human DNA has no effect on NIH/3T3 cells. The coding regions of the normal N‐ras gene have been sequenced and the predicted amino acid sequence of the N‐ras product is very similar to that of the c‐Ha‐ras1 and c‐Ki‐ras2 products. By making chimeric molecules between the two cloned genes the activating alteration in the HT1080 N‐ras gene has been localised to a single base change that results in an amino acid alteration at position 61 of the p21 N‐ras product.

The primary amino acid structures of the 43-kDa (A) and 15-kDa (B) subunits of the 58-kDa form of the hormone inhibin have been elucidated by cloning and analysis of cDNA species derived from bovine granulosa cell mRNA.The A subunit (Mr = 32,298) is a protein of 300 amino acids with two potential N-glycosylation sites and two potential proteolytic processing sites and'has a pre-pro region of 60 amino acids. The mature B sublinit (Mr = 12,977) is a protein of 116 amino acids synthesized from a separate mRNA. These data establish that a 31-kDa forn of inhibin also isolated from bovine follicular fluid, with subunits qf 20 kJa (Ac) and 15 kDa (B), is derived from the 58-kDa form by proteolytic processing of the A subunit.Considerable evidence has now accumulated to support the concept that the gonads produce a protein termed inhibin (1) that selectively suppresses the pituitary secretion of folliclestimulating hormone (2), a key hormone in controlling folliculogenesis and spermatogenesis. Controversy still exists concerning the nature of inhibin (3) partly because of the fact that some of the bioassays used in its characterization do not monitor follicle-stimulating hormone directly (4). For instance, inhibin-like activity has been detected in seminal plasma; the proteins associated with this activity have been purified and characterized but subsequently they were shown not to be of gonadal origin (5)(6)(7)(8)(9).We have isolated inhibin from a gonadal source, bovine follicular fluid, with an apparent molecular mass of 58 kDa, composed of two subunits now designated A and B, of 43 kDa and 15 kDa, respectively, linked together by disulfide bonds (10). Subsequently, 32-kDa inhibin was isolated by others from porcine follicular fluid, with two subunits of 18/20 kDa and 13/14 kDa (11,12). We have also shown that a 31-kDa form of bFF inhibin, composed of 20-kDa and 15-kDa subunits, is generated during a pH precipitation step in the purification procedure (13) but the precise relationship between the different forms has been unknown. However, the biological activity of the 31-kDa form is neutralized in vitro by an antiserum raised against the 58-kDa form (13), suggesting that the 31-kDa inhibin is a processed form of the 58-kDa inhibin.This article describes the amino acid sequences of the two subunits of the 58-kDa bovine inhibin as determined fromn cDNA sequencing and reports that the 20-kDa subunit of the 31-kDa inhibin is derived from the 43-kDa subunit of the 58-kDa inhibin, thus clarifying the relationship between these different forms.MATERIALS AND MIETIODS NH2-Terminal Amino Acid Sequencing of the 58-kDa Inhibin. The 58-kDa inhibin was isolated from bFF (10).Briefly, this involved a four-step' purjfication procedure: (i) gel permeation chromatography on Sephacryl 3200 in 0.05 M ammonium acetate, (it) gel permeation chromatography on Sephadex G100 in 4 M acetic acid, (iii) reversed-phase HPLC on an Ultrapore RPSC column-(Beckman) using a 0.1% trifluoroacetic acid-acetonitrile-H2O gradient, and (iv) prepara...

The structure and organisation of the human N-ras gene has been determined by analysing cDNA clones derived from the two main mRNA transcripts. One clone in particular is 4.1 Kb long and originates from the larger (4.3 Kb) message. Sequence analysis of this clone has revealed that the N-ras gene consists of seven exons. A second clone deriving from the smaller (2 Kb) message shows that the difference between the two transcripts is a simple extension through the termination site of the 2 Kb transcript. Using S1 analysis, two transcriptional starts have been mapped, 10 bp apart. There is no obvious TATA box in the expected promoter region of the gene, though there are 4 GGGCGG sequences surrounding the start sites. The 5' untranslated sequence contains 2 ATGs upstream of the initiation codon.

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Seven Merino-Border Leicester cross-bred ewes were immunized with a purified fusion protein, produced by recombinant DNA methods, of the alpha subunit of bovine inhibin. Four animals were immunized with the fusion protein alone and three with a conjugate made by coupling the fusion protein to keyhole limpet haemocyanin (KLH) using glutaraldehyde. Each animal received four injections of the fusion protein over 93 days. The animals were synchronized using progestagen sponges and subjected to laparoscopy for the determination of ovulation rates in two consecutive cycles (days 115 and 135). The immunized animals had overall mean ovulation rates for each cycle of 3.4 and 3.4 which was significantly (P less than 0.001) above the rates of 1.1 and 1.4 determined for the controls, which had either received no treatment (n = 5) or had been immunized with 300 micrograms KLH (n = 4). Analysis of antisera taken on day 115 showed significant fusion protein antibodies and iodinated inhibin-binding capacity in the test but not control groups. Furthermore, antisera to the fusion protein in four out of seven ewes neutralized the inhibin bioactivity of ovine follicular fluid in an in-vitro bioassay. These data demonstrate that neutralization of inhibin can be effected by immunization with bovine inhibin alpha subunit and that such immunization results in increased ovulation rates as predicted from the biological role of inhibin as a suppressor of FSH.