Biomedical subjects

R M Gubits

Publications and source records attributed to R M Gubits.

23 records · Page 2Linked to original sources

PubMed1985

In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse.

Epidermal growth factor (EGF) is a polypeptide originally isolated from the mouse submandibular gland, where it is localized immunocytochemically in cells of the granular convoluted tubules (GCT). cDNAs encoding the precursor of mouse submandibular EGF have been cloned (Scott et al. Science 221:236, 1983; Gray et al. Nature 303:722, 1983). A fragment of one of these clones, pmegf10, containing the EGF coding region, was tritium-labeled by nick-translation and used as a probe for in situ hybridization to EGF mRNA. A specific hybridization signal for EGF mRNA was seen only in mature or developing GCT cells. The intensity of the signal was stronger in glands of intact males than in females or in castrated males. In glands of castrates treated with testosterone, or of intact females treated with triiodothyronine (T3), the signal was comparable to that in intact males. In glands of males treated with T3 the intensity of the signal was stronger than in untreated males. A weak to moderate signal was seen in developing GCT cells of 20-day-old males but not females. Hybridization for 3 days gave a stronger signal than that for 1 day. No signal was seen in either sex at 10 days of age, or in control preparations exposed to labeled DNA of pBR322. The presence of EGF mRNA exclusively in GCT cells provides strong evidence that these cells are the only site of synthesis of EGF in the submandibular gland. In situ hybridization with this cDNA probe will provide a sensitive method to determine possible cellular sites of EGF production outside of the submandibular gland.

Animals↗

PubMed1984

Fast skeletal muscle myosin light chains 1 and 3 are produced from a single gene by a combined process of differential RNA transcription and splicing.

Fast myosin light chains (MLCf) 1 and 3 are proteins associated with the thick filament in vertebrate fast muscle fibers. MLC1f and MLC3f have complete sequence homology for the first 141 amino acids from their COO- end, but they differ in length and amino acid sequence at their NH+3 ends (MLC1f = 49 amino acids, MLC3f = 8 amino acids), and they are translated from different mRNAs. To elucidate the structural relationship between mammalian fast myosin light chain 1 and 3 isoforms, cDNA clones have been isolated from rat skeletal muscle and their primary nucleotide sequences were determined. MLC1f and MLC3f mRNAs display complete sequence identity in the 3' untranslated sequences (285 base pairs) and in the codons specifying the 142 carboxyl-terminal amino acids. In contrast, the sequences encoding the amino-terminal 49 and 8 amino acids of MLC1f and MLC3f, respectively, as well as their 5' untranslated regions, are highly divergent. Using the cloned cDNAs as probes we have isolated the single gene locus encoding both MLC1f and MLC3f in four overlapping genomic clones spanning over approximately 25 kilobase pairs (kb) of DNA. The sequences encoding the common body of MLC1f and MLC3f are distributed in 4 separate exons at the 3' end of the gene. The start of transcription site and 5' untranslated region of MLC3f is found 5 kb upstream from the common body while the MLC1f mRNA transcription start site and the exon specifying the 5' untranslated region and amino acid 1-40 of MLC1f lies about 10 kb further upstream. Strikingly, a mini-exon that specifies amino acids 41-49 of MLC1f is located downstream from the two MLC3f-specific exons (5' untranslated and amino acids 1-8) and is separated from the upstream MLC1f exons by 12 kb of DNA. This bizarre gene organization implies a novel form of alternative promoter utilization and RNA splicing that is tissue-specific and developmentally regulated in order to generate the mature MLC1f and MLC3f mRNAs from a single gene.

Amino Acid Sequence↗

PubMed1984

Differential regulation of alpha 2u globulin gene expression in liver, lachrymal gland, and salivary gland.

The alpha 2u globulins, products of a highly homologous multigene family, are synthesized in the liver and submaxillary salivary glands of the rat. Although their precise function has not been ascertained, they are of interest because of the complex developmental and hormonal regulation of their tissue levels. We now report that alpha 2u globulin is synthesized in a third tissue of the rat, the extraorbital lachrymal gland. Immunocytochemical studies indicate that the distribution of alpha 2u globulin is more homogeneous in the lachrymal gland than in the liver or submaxillary gland. In situ hybridization to alpha 2u globulin RNA reveals specific signal only over the acinar cells of the lachrymal gland. Several different isoelectric forms of alpha 2u globulin are encoded by lachrymal gland mRNA. The major lachrymal and salivary gland isoforms are indistinguishable from one another, but more acidic than the hepatic isoforms. In addition, analysis of double-stranded cDNAs with a diagnostic restriction-enzyme pair detects no differences between the alpha 2u globulin mRNAs of lachrymal and salivary gland, but clearly distinguishes these from their hepatic counterparts. In spite of the similarity between the lachrymal and salivary gland alpha 2u globulin gene products, we find that the hormonal and developmental regulation of alpha 2u globulin expression differs markedly in these two tissues. In the liver, where a different subset of alpha 2u globulin genes is expressed, a third regulatory phenotype is observed.

Alpha-Globulins↗

PubMed1983

Characterization of sarcomeric myosin heavy chain genes.

Myosin heavy chain is encoded by a large multigene family. Using pMHC-25, a recombinant cDNA clone isolated from the rat myogenic cell line L6E9, four members of this family in the rat have been isolated and shown to be tissue-specific and developmentally regulated. The coding regions of these genes share regions of homology interspaced with regions of non-homology. Detailed analysis of one embryonic and one adult myosin heavy chain gene shows that the coding sequences are interrupted by numerous intervening sequences whose number, size, and distribution do not appear to be conserved in the same organism or between species.

Animals↗

PubMed1982

Sarcomeric myosin heavy chain is coded by a highly conserved multigene family.

pMHC25, a recombinant plasmid containing myosin heavy chain (MHC) cDNA sequences from differentiated myotubes of the L6E9 rat cell line, has been shown to hybridize to all sarcomeric MHC mRNAs so far tested but not to nonsarcomeric MHC mRNAs. In addition, pMHC25 hybridizes to multiple restriction endonuclease-digested fragments of rat genomic DNA corresponding to different MHC genomic sequences. Thus, the MHC gene represented by pMHC25 is a member of a sarcomeric MHC multigene family that has regions of sequence homology shared among its members. This sarcomeric MHC multigene family has been estimated to be composed of a minimum of seven genes, some of which are polymorphic in the rat. We have also determined that pMHC25 hybridizes to MHC gene sequences in genomic DNA of all species that have striated muscle, ranging from nematodes to man. Sarcomeric MHC genes, therefore, have been horizontally and vertically conserved in evolution. Additionally, we have used the pMHC25 plasmid to demonstrate that MHC genes do not undergo rearrangement or amplification during muscle cell differentiation.

Animals↗