Biomedical subjects

J C Groppe

Publications and source records attributed to J C Groppe.

6 recordsLinked to original sources

PubMed2001

Expression, purification, and characterization of a soluble form of the first extracellular domain of the human type 1 corticotropin releasing factor receptor.

The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.

Amino Acid Sequence↗

PubMed1995

Sequence-independent detection of gene family homologs: identification of a transcript encoding a molluscan serine protease homologous to the pancreatic enzymes of vertebrates.

Autoradiography of 32P-labeled cDNA, fractionated at high resolution by electrophoresis through thin (0.8-1.5 mm) vertical alkaline agarose gels, provides a sequence-independent screening procedure for gene family homologs. A screen of tissues of a marine mollusc revealed a prominent intestine-specific cDNA encoding a pancreatic serine protease homolog, which was not detectable as a discrete poly(A)+ RNA species on formaldehyde agarose gels. Discrete cDNA products are authentic, non-truncated transcripts of tissue-specific mRNA. A band-sharpening effect is imparted to cDNA products due to (a) substitution of a uniform length 5'-oligo(dT) terminus for heterogeneous 3'-poly(A) termini and (b) the inherent superior resolution of alkaline-denatured DNA.

Animals↗

PubMed1993

Molluscan chymotrypsin-like protease: structure, localization, and substrate specificity.

A messenger RNA encoding a chymotrypsin-like preproprotease is expressed abundantly and specifically in the distal quarter of the intestine of the mollusc Haliotis rufescens (red abalone). Consistent with this finding, a chymotrypsin-like activity was detected at highest concentration in the lumen of this segment of the intestine. Because the complexity of total protein in the distal intestinal fluid was low and the chymotrypsin-like protease was highly expressed, purification of the enzyme to near homogeneity was achieved by a single passage over an anion-exchange resin. The primary specificity of the protease, predicted from homology of the key amino acid residues (Ser189, Gly216, and Ser226) in the substrate binding site with similar residues lining the S1 subsites of other chymotrypsin-like enzymes, was confirmed by hydrolysis of a family of tetrapeptide substrates with different P1 amino acids. The optimal P1 residues include those with bulky, gamma-branched side chains (phenylalanine and leucine) similar to the side chain of the asparagine residue at P1 of the activation peptide of the proenzyme. Thus, unlike zymogens of the pancreatic serine proteases, which are activated by the common tryptic mechanism, the zymogen of the molluscan enzyme appears to be activated by an autocatalytic mechanism, i.e., by cleavage with active chymotrypsin-like protease. Additional unique properties of the enzyme predicted from the primary sequence include an unpaired cysteine, with the potential for internal thiol-disulfide isomerization between two different conformers of the protease.

Amino Acid Sequence↗

PubMed1993

Isolation of full-length RNA templates for reverse transcription from tissues rich in RNase and proteoglycans.

RNA isolated by conventional guanidinium isothiocyanate methods from tissues of a mollusc (red abalone: Haliotis rufescens) is largely degraded and discolored by contaminants. These contaminants are associated with inhibition of reverse transcriptase, prevent accurate spectrophotometric determination of RNA concentration, and impart undesirable viscosity to the preparations. A cold two-step method of RNA isolation was devised which provides high yields of full-length RNA templates from these tissues and eliminates the discolored contaminant. Immediately following homogenization of tissues at ca. 5 degrees C, which proved crucial for the recovery of high-molecular weight species, the RNA is isolated from the bulk of the RNase by a single acid-phenol-chloroform extraction at 0 degrees C. The inhibitor of reverse transcriptase, suspected to be a proteoglycan (or a similar high-molecular-weight polyanion) component of the intestinal mucus, is eliminated only by a second purification step employing ultracentrifugation through a dense cushion of CsCl. This cold two-step method should prove useful for providing full-length RNA templates relatively free of polysaccharide, a common contaminant of RNA preparations, from both plant and animal tissues.

Amino Acid Sequence↗

PubMed1982

Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

Base Sequence↗

PubMed1981

Construction and characterization of three yeast-Escherichia coli shuttle vectors designed for rapid subcloning of yeast genes on small DNA fragments.

We have constructed three new subcloning plasmid vectors, pRC1, pRC2, and pRC3, derived from pKC7, which allow the rapid, single-step subcloning of yeast genes. Subcloning with these vectors utilizes a partial digestion with Sau3A to generate a quasi-random set of DNA fragments from the original plasmid. All three vectors contain a kanamycin resistance gene. Therefore, if the original cloned yeast DNA fragment is present in a vector that does not specify kanamycin resistance, the subclone pool can be propagated in Escherichia coli in the presence of kanamycin to select against parent plasmids that escaped restriction by Sau3A. Selection by complementation in yeast yields a collection of plasmids with smaller yeast DNA inserts containing the gene of interest. In the vectors pRC2 and pRC3, constructed from pRC1, the unique BamHI site is located within an intact tetracycline resistance gene, thus making it possible to screen bacterial transformants for those containing recombinant plasmid molecules. Vectors pRC2 and pRC3 also contain the yeast 2 micrometers DNA replication origin, and thus are more stable than plasmids carrying only the TRP1-associated replicator (ars1).

Cloning, Molecular↗