John M. Chirgwin

46 kd mannose 6-phosphate receptor: Cloning, expression, and homology to the 215 kd mannose 6-phosphate receptor

We have isolated cDNA clones encoding the entire sequence of the bovine 46 kd cation-dependent mannose 6-phosphate (CD Man-6-P) receptor. Translation of CD Man-6-P receptor mRNA in Xenopus laevis oocytes results in a protein that binds specifically to phosphomannan-Sepharose, thus demonstrating that our cDNA clones encode a functional receptor. The deduced 279 amino acid sequence reveals a single polypeptide chain that contains a putative signal sequence and a transmembrane domain. Trypsin digestion of microsomal membranes containing the receptor and the location of the five potential N-linked glycosylation sites indicate that the receptor is a transmembrane protein with an extracytoplasmic amino terminus. This extracytoplasmic domain is homologous to the approximately 145 amino acid long repeating domains present in the 215 kd cation-independent Man-6-P receptor.

Evaluation of rat insulin messenger RNA in pancreatic and extrapancreatic tissues.

SummaryThe purpose of these studies was to determine whether insulin detected immunochemically in extrapancreatic tissues of the adult rat is synthesized in situ by quantitating mRNA in these tissues. A blot hybridization assay was utilized with cloned 32P-proinsulin cDNA. The lower limit of detection was estimated to be 3pg. Proinsulin mRNA concentration was found to be 1000–1500 μg in isolated pancreatic islets and was easily detected in total pancreatic RNA at 10–15 pg/ μg. Proinsulin mRNA was quantitated in rat insulinoma cells adapted to culture at levels 1∶50 those in normal islets. Samples of RNA (20–50 μg) enriched about 50-fold for mRNA sequences by repeated oligo-deoxythymidylate chromatography were assayed. No insulin mRNA was detected in 50 μg samples of RNA from brain or in 20 μg samples from subsections of brain or other extrapancreatic tissues. RNA samples were undegraded as assessed by ability to stimulate protein synthesis in a cell-free system. Proinsulin mRNA from pancreas was added to brain homogenates and recovered intact. Brain RNA samples with insulin mRNA levels 1:1000 that of pancreas would be predicted to have 50–75 pg proinsulin mRNA/50 μg sample assayed if present. Because none was found, brain must have a concentration <1:6,000 that of pancreas. These findings suggest that immunoassayable insulin detected in extrapancreatic tissues of the adult rat is synthesized by the pancreas.

Human renin gene is on chromosome 1

DNA sequences encoding kidney renin were localized to region p21 → qter of human chromosome 1 by Southern blot analysis of mouse-human somatic cell hybrids with a cloned human renin DNA probe. The renin gene may be a member of a chromosome 1 linkage group which is conserved in mouse and man. Available evidence suggests this gene is present in one copy per haploid genome. Thus those renin-like molecules detected immunologically in tissues other than the kidney (such as brain, placenta, uterus, pituitary, vasculature, and adrenal) may be derived from this single gene. Since renin messenger RNA in human kidney is about 1550 nucleotides long, reported molecular weights in excess of 45,000 for circulating renin represent posttranslational or postsecretory modifications of the polypeptide.

Mouse Kidney Renin Gene Is on Chromosome One

The structural gene for mouse kidney renin (Ren-1)was localized to chromosome 1 by Southern blot analysis of mouse-hamster somatic cell hybrids with a cloned mouse submandibular renin cDNA probe. The submandibular renin gene (Ren-2)also lies on chromosome 1; so it may be, in those mouse lines which carry it, a tandem duplication of the kidney-type Ren-1gene.

Insulin Gene Structure and Function: A Review of Studies Using Recombinant DNA Methodology

This review focuses on recent advances in molecular biology as they pertain to the insulin gene and diabetes mellitus. The structure of the human insulin gene is examined, andfactors related to its normal functioning in the beta cells of the pancreas are explored.DNA polymorphisms near the insulin locus and their relationship with certain types of diabetes are considered, as are recently characterized human insulin gene mutations. Events in animal models for diabetes that reflect altered insulin gene expression are discussed and the potential application of gene therapy in human diabetes is examined. Recombinant DNA methodology holds great promise as a tool for providing better understanding of the causes of diabetes and potential curative treatment.

Analysis of insulin gene expression in human pancreas

The purpose of these studies was to determine whether insulin gene expression at the level of proin-sulin mRNA could be studied in human pancreas. RNA was isolated from autopsy specimens and analyzed by RNA-blot hybridization with various 32P-human insulin gene probes spanning either the entire gene or the second intervening sequence. A major band 0.62 kilo-bases (kb) in length accounted for over 95% of the mRNA, consistent in size with presumed mature proin-sulin mRNA. In addition, minor bands of 1.5 and 1.3 kb were seen, consistent with an initial gene transcript containing both intervening sequences and with a processed intermediate. The 1.5-and 1.3-kb RNA were confirmed to be proinsulin mRNA precursors by hybridization specifically with the IVS II probe. Total RNA and polyadenylated RNA from five normal pancreata and two insulinomas revealed the same pattern. This method provides a means of determining whether altered insulin gene expression is one cause of diabetes.