Peter H. Seeburg

Structure of two human ovarian inhibins

The complete amino acid sequences of two forms of human ovarian inhibin have been determined through cloning and nucleotide sequencing of cDNAs encoding their individual subunit precursors. The alpha subunit common to both forms of human inhibin is homologous (84 percent conserved) to its equivalent porcine alpha subunit; the subunits which are different in both inhibins (beta A and beta B) are identical to their porcine equivalents in all but one of 232 sequence positions.

Alpha subunit variants of the human glycine receptor: primary structures, functional expression and chromosomal localization of the corresponding genes.

Two cDNAs encoding variants (alpha 1 and alpha 2) of the strychnine binding subunit of the inhibitory glycine receptor (GlyR) were isolated from a human fetal brain cDNA library. The predicted amino acid sequences exhibit approximately 99% and approximately 76% identity to the previously characterized rat 48 kd polypeptide. Heterologous expression of the human alpha 1 and alpha 2 subunits in Xenopus oocytes resulted in the formation of glycine‐gated strychnine‐sensitive chloride channels, indicating that both polypeptides can form functional GlyRs. Using a panel of rodent‐human hybrid cell lines, the gene encoding alpha 2 was mapped to the short arm (Xp21.2‐p22.1) of the human X chromosome. In contrast, the alpha 1 subunit gene is autosomally located. These data indicate molecular heterogeneity of the human GlyR at the level of alpha subunit genes.

Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase)

We have isolated a cDNA clone encoding human enkephalinase (neutral endopeptidase, EC 3.4.24.11) in a λgt10 library from human placenta, and present the complete 742 amino acid sequence of human enkephalinase. The human enzyme displays a high homology with rat and rabbit enkephalinase. Like the rat and rabbit enzyme, human enkephalinase contains a single N‐terminal transmembrane region and is likely to be inserted through cell membranes with the majority of protein, including its carboxy‐terminus, located extracellularly.

The Mammalian GnRH Gene and Its Pivotal Role in Reproduction

Publisher Summary The key regulatory brain peptide controlling reproduction in mammals and sub-mammalian vertebrate species is the decapeptide luteinizing hormone-releasing hormone (LHRH), also called gonadotropin-releasing hormone (GnRH). This peptide, synthesized in and secreted from hypothalamic neurosecretory cells, stimulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from pituitary gonadotrophs. The miniscule amounts of GnRH in hypothalamus as well as its modified N- and C-termini made its isolation and structural determination a monumental task. This chapter explains the structure and restriction map of the human and rat GnRH-GAP gene. The description of the gene structure and the GnRH precursor sequence has aided in the investigation of GnRH gene expression in various parts of the mammalian organism. Using mainly immunological procedures, it has been found that GAP and GnRH are produced in a surprisingly large number of tissues. They occur in varying amounts in placenta, mammary tissue, gonads, kidney, as well as in certain extra-hypothalamic parts of the central nervous system.

Mapping of genes for inhibin subunits α, βA, and βB on human and mouse chromosomes and studies of jsd mice

Abstract Inhibin (INH) is a gonadal glycoprotein hormone that regulates pituitary FSH secretion and may also play a role in the regulation of androgen biosynthesis. There are two forms of inhibin that strongly inhibit pituitary FSH secretion. These share the same α subunit that is covalently linked to one of two distinct β subunits ( β A or β B . However, dimers of two β subunits are potent stimulators of FSH synthesis and release in vitro . The β subunits share extensive sequence similarity with transforming growth factor β. Recently isolated cDNAs for all three inhibin subunits have been used to map their cognate loci on human and mouse chromosomes by Southern blot analysis of somatic cell hybrid DNAs and by in situ hybridization. INHα and INH β B genes were assigned to human chromosome 2, regions q33 → qter and cen → q13, respectively, and to mouse chromosome 1. The INH β A locus was mapped to human chromosome 7p15 → p14 and mouse chromosome 13. The region of mouse chromosome 1 that carries other genes known to have homologs on human chromosome 2q includes the jsd locus (for juvenile spermatogonial depletion). Adult jsd jsd mice have elevated levels of serum FSH and their testes are devoid of spermatogonial cells. The possibility that the mutation in jsd involves the INHα or INH β B gene was investigated by Southern blotting of DNA from jsd jsd mice, and no major deletions or rearrangements were detected.

Human Luteinizing hormone-releasing hormone gene (LHRH) is located on short arm of chromosome 8 (region 8p11.2 → p21)

Luteinizing hormone-releasing hormone (LHRH) is synthesized by hypothalamic neurons and affects the release of gonadotropic hormones from the anterior pituitary gland. A cDNA clone encoding the human LHRH precursor molecule was used to assign theLHRH gene to a human chromosome by in situ hybridization and Southern blot analysis. Metaphase spreads from two normal individuals were hybridized with3H -labeled LHRH-specific sequence. Of 120 cells analyzed, 33 had silver grains over the p11.2 → p21 bands of chromosome 8. No other chromosomal site was labeled above background, indicating the presence of a single site for LHRH sequences in the human genome. Independent confirmation for this location of the humanLHRH gene on chromosome 8p was provided by analysis of DNA from human × Chinese hamster somatic cell hybrids. DNA samples were digested with EcoRI, blotted, and hybridized with the32P-labeled human LHRH precursor cDNA probe. The single 11.5-kb human-specific band was detected only in hybrids containing human chromosome 8. Also, hybridization was observed in DNA from hybrids in which a portion of human chromosome 8 (region 8pter → 8q21) had been spontaneously translocated onto a Chinese hamster chromosome.

The proopiocortin (adrenocorticotropin/β-lipotropin) gene is located on chromosome 2 in humans

The proopiocortin gene is located on chromosome 2 in humans. A 13-kb DNA fragment containing proopiocortin gene sequences was identified in human cells while proopiocortin-related gene sequences of 9.8 and 6.2 kb were present in mouse cells. In human-mouse cell hybrids which contained reduced numbers of human chromosomes and a complete set of mouse chromosomes, the 9.8- and 6.2-kb fragments were always present while the 13-kb fragment segregated with human chromosome 2 and the chromosome 2 enzyme markers acid phosphatase-1 (ACP1), malate dehydrogenase-1 (MDH1), and isocitrate dehydrogenase-1 (IDH1). Analysis of a single cell hybrid with a broken chromosome 2 indicates that the proopiocortin andACP1 genes are closely linked and in the distal region of the short arm of chromosome 2.

Molecular Forms of Gonadotropin-Releasing Hormone Associated Peptide (GAP): Changes within the Rat Hypothalamus and Release from Hypothalamic Cells in vitro

We have developed RIAs using antisera directed against the cryptic peptide of the GnRH precursor (termed GnRH-associated peptide, GAP) and have used these together with a GnRH assay to characterize proGnRH-derived peptides in rat hypothalamic extracts. On Sephadex chromatography we have identified three molecular forms of GAP-like immunoreactivity (GAP-LI), in addition to the GnRH decapeptide. The largest of these forms is an 8.0-kilodalton (kD) GAP-LI which appears to be the complete proGnRH peptide. The second is a 6.5-kD GAP-LI, and is similar to the complete cryptic peptide (i.e. proGnRH14-69 or GAP1.56). The third peptide is a 2.5 kD C-terminal fragment of the cryptic peptide, representing a processed form of GAP. In whole hypothalamic extracts from normal rats the 8.0-kD form was the major form, comprising 60-70% of the total GAP-LI. All three forms were present in three distinct areas of the rat hypothalamus, namely median eminence (ME), anterior and mid-hypothalamus. However in the ME the proportion of 8.0-kD GAP-LI was significantly reduced and the proportion of 6.5-kD GAP-LI significantly increased compared to anterior and mid-hypothalamic samples (p less than 0.05). In whole hypothalamic extracts from pregnant and lactating rats the total content of proGnRH-derived peptides was reduced but the relative proportions of these peptides were not significantly changed from normal female rats. However, in postlactating rats, 2 weeks after removal of pups, the total levels of GAP-LI were unchanged compared to normals, but the percentage of 8.0-kD GAP-LI was significantly decreased and the percentage of 2.5-kD GAP-LI significantly increased compared to normals (p less than 0.05), suggesting that proGnRH may undergo additional processing dependent on physiological condition. In fetal and neonatal rats the proportion of the 6.5-kD peptide was increased and that of the 8.0-kD peptide decreased compared to adults, and this change became less significant with increasing age. In ovariectomized rats the proportion of 6.5-kD GAP-LI was increased and that of 8.0-kD GAP-LI decreased; this change was partially reversed with steroid treatment. Both the 6.5 and 2.5-kD forms were released by high K+ stimulation of neonatal hypothalamic cells in culture. These results indicate that there is differential processing of the proGnRH precursor within the hypothalamus and in altered physiological states.

Gonadal development and gametogenesis in the hypogonadal mouse are restored by gene transfer.

These results describe controlled regulation of a mammalian neural gene in transgenic mice. Analysis of truncated GnRH-GAP genes in transgenic mice will enable us to define the DNA sequences responsible for this control. Furthermore, by separate mutation of the GnRH and GAP coding sequences we will be able to determine the relative importance of these two peptides in the development and maintenance of reproductive function.

Are Activated Proto-onc Genes Cancer Genes?

The main objective of cancer molecu~ar biology is to identity cancer genes. ~espite fierce efforts, this objective has still not been met [1-3]. As yet the only known cancer genes are the transformin~ one g~n.e~ of retroviruses. Typically these VIruses InItiate and maintain cancers with autonomous transforming genes that ~re domina~t in susceptible cells [5]. The dIsc~very of ~Ingle gene determinants of cancer In retroVIruses has become a precedent that has infected cancer gene research. It has made retroviral one genes the favorite models of cellular oncogenes, although th~ releva~ce of single-gene models to VIrus-negative tumors is as yet unknown. Fortunately, one genes are either detrimental or at least useless to the viability of the virus and thus are not maintained by retroviruses. They are the products of rare, genetic ac.cid~nts, generated by illegitimate recombmatIOns between retroviruses and cellular genes,