A new member of the insulin receptor family, insulin receptor-related receptor, is expressed preferentially in the kidney.

Shier and Watt isolated human and guinea pig genomic DNA encoding a putative protein, insulin receptor-related receptor (IRR), the primary structure of which is similar to that of other members of the insulin receptor family, the insulin receptor and type-I IGF receptor (J. Biol. Chem. 264, 14605-14608 (1989)). However, the expression of the IRR gene remained unknown. In this paper, we isolated the IRR cDNA from the rat brain and examined the expression of the IRR mRNA in a variety of rat tissues, including the brain, heart, lung, liver, small intestine, kidney, thymus, spleen, muscle, adipose tissue and cartilage by polymerase chain reaction. In contrast to the wide distribution of the insulin receptor and type-I IGF receptor mRNAs, the IRR mRNA is expressed preferentially in the kidney, which indicates that IRR has unique functions as a member of the insulin receptor family.     

Cloning and sequencing of the complete cDNA encoding the human insulin receptor related receptor.

The insulin receptor related receptor (IRR) is a heterotetrameric transmembrane receptor with intrinsic tyrosine kinase activity. The IRR shares large homology with the insulin and the insulin-like growth factor-1 (IGF-I) receptor with regard to amino acid sequence and protein structure. So far, only a partial human sequence containing the complete 3' end has been reported, although the full-length human IRR cDNA had been used for transfection studies and functional analysis of the receptor. We have isolated a full-length human IRR cDNA and report on the 5' translated and untranslated region of the human IRR gene. The full length IRR sequence contains 4150 bases and shares a high degree of homology with the guinea pig IRR cDNA sequence and rat IRR sequences that had been reported earlier on by others. Sequencing of the IRR cDNA revealed that the human IRR cDNA contains 341 bases corresponding to the IRR 5' end in addition to the bases that had been reported on before. Also, this sequence contains the start codon of translation. The full length cDNA for the human IRR can now be used for functional expression studies and to elucidate the nature of the ligand for this receptor type.     

Immunological identification of rat tissue kallikrein cDNA and characterization of the kallikrein gene family

A tissue kallikrein cDNA was identified by direct immunological screening with affinity-purified anti-rat tissue kallikrein antibody from a rat submandibular cDNA library constructed with the expression vector pUC8. Sequence analysis of the kallikrein cDNA revealed an encoded protein 97% homologous to the partial amino acid sequence of rat submandibular kallikrein. This cDNA was used to hybrid-select kallikrein-specific RNA from submandibular gland. Translation of the hybrid-selected RNA in a cell-free assay system resulted in the production of a 37 kDa peptide representing the preproenzyme. In addition, hybrid-selection of RNA under less stringent conditions showed cross-hybridization with other submandibular gland mRNA species. In correlation with these results, analysis of rat genomic DNA showed extensive hybridization, suggesting a family of closely related kallikrein-like genes. Consequently, a Charon 4A rat genomic library was screened for kallikrein genes by hybridization with rat tissue kallikrein cDNA. Thirty-four clones were isolated and found to be highly homologous by hybridization and restriction enzymes analyses. Fourteen unique clones were identified by restriction enzyme site polymorphisms within DNA segments which hybridized to the kallikrein cDNA probe and it was estimated that at least 17 different kallikrein-like genes are present in the rat. Sequence and structural analysis of one of the genomic clones revealed a gene structure similar to that of other serine proteinases. Comparison of the partially sequenced exon regions of the gene with the sequence of rat tissue kallikrein cDNA reveals 89% identity when aligned for the greatest homology. However, the genomic sequence predicts termination codons in all three translational reading frames, implying that this gene is nonfunctional, i.e., a pseudogene. Comparison of the rat genomic sequence to a kallikrein-like gene from the mouse reveals extensive preservation of exons, less identity within introns and no significant homology between extragenic regions.     

A frameshift mutation destabilizes androgen receptor messenger RNA in the Tfm mouse.

A composite mouse androgen receptor DNA sequence was obtained by amplifying genomic DNA or cDNA using the polymerase chain reaction. The open reading frame was 2,697 basepairs, encoding a polypeptide of 899 amino acids (98,204 mol wt). Amino acid sequence comparisons indicated that the mouse androgen receptor (AR) is 97% homologous with rat AR and 83% with human AR. The amino acid sequences of the three receptors are identical within the DNA- and steroid-binding domains. Northern blot analysis revealed the predominant mouse AR mRNA to be 10 kilobases (kb). A 1.7-kb mRNA species was detected in mouse kidney using a cDNA probe containing only 5' untranslated AR sequence. Lack of hybridization with AR-coding sequence probes suggested that the 1.7-kb mRNA was not a truncated form of AR mRNA. Sequencing of genomic DNA isolated from testicular feminized (Tfm) mice revealed a single base deletion in the N-terminal domain, resulting in a frameshift mutation. Cycloheximide treatment caused a dramatic increase in AR mRNA in kidneys of Tfm mice, but not wild-type mice, suggesting that the Tfm mutation results in an unstable AR mRNA.     

Localization of the insulin receptor-related receptor gene to human chromosome 1

DNA encoding the insulin receptor-related receptor (IRR), a novel receptor whose predicted primary structure is similar to those of the insulin and insulin-like growth factor I receptors, has been used in Southern blot analysis of DNA from human x mouse somatic cell hybrids to assign the IRR gene (INSRR) to human chromosome 1.     

Structure and expression of human and rat D2 dopamine receptor genes.

D2 dopamine receptor may be related with the pathogenesis of Parkinson's disease and schizophrenia. Furthermore, the antipsychotic drugs have high affinity for D2 dopamine receptor. We carried out the cloning of the genomic DNA for human D2 dopamine receptor and clarified the structure of this gene. Our isolated gene spans about 15 kbp and consists of seven exons interrupted by six introns. However, putative first exon was not yet identified. Spot blot hybridization analysis of cell sorter fractionated human chromosomal DNA with D2 receptor genomic DNA revealed the localization of this gene in the chromosome 11 fraction. We analyzed human genomic DNA by Southern blot hybridization with D2 dopamine receptor genomic DNA as a probe, but so far we could not find RFLP. Northern blot analyses of brain RNA of several animals and rat brain RNA after various treatments were carried out. Developmental changes of D2 dopamine receptor mRNA were observed in the rat brains.     

The plant ferredoxin precursor: nucleotide sequence of a full length cDNA clone.

A cDNA clone (pFD1) derived from Silene pratensis ferredoxin mRNA was selected from a cDNA-library using the hybrid released translation technique. Nucleotide sequence analysis showed the cDNA insert to contain the complete coding region of the ferredoxin precursor protein. The ferredoxin precursor has a mol.wt. of 15 300, the transit-peptide has a mol.wt. of 5600. The length of the ferredoxin mRNA was found to be 700 nucleotides whereas the cDNA insert was about 1200 basepairs. S1 nuclease protection experiments showed the ferredoxin-specific DNA to be 660 basepairs in length and to start 39 nucleotides upstream of the ferredoxin coding sequence. Southern blot analysis of genomic DNA revealed the presence of only one fragment with homology to the ferredoxin cDNA probe, so it is probably a single-copy gene. Comparison of the ferredoxin transit-sequence with transit sequences of another stromal protein, the small subunit of ribulosebisphosphate carboxylase showed no apparent homology, except for a stretch of three amino acids near the processing site.     

Molecular cloning of the human insulin gene

Isolation of genomic clones containing the human insulin gene by screening the human genomic library for this gene using the cDNA rat insulin probe is reported. The analysis of promoter-enhancer region and exons sequences has revealed their identity to analogous sequences determined earlier.     

Human gastric intrinsic factor: characterization of cDNA and genomic clones and localization to human chromosome 11

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5' untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3-10 encode the remainder of the coding and 3' noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human X mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.     

Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland.

Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning. Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody. Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments. Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides. Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat. Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement. Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor. Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.     

Structure and localization of genes encoding aberrant and normal epidermal growth factor receptor RNAs from A431 human carcinoma cells.

A431 cells have an amplification of the epidermal growth factor (EGF) receptor gene, the cellular homolog of the v-erb B oncogene, and overproduce an aberrant 2.9-kilobase RNA that encodes a portion of the EGF receptor. A cDNA (pE15) for the aberrant RNA was cloned, sequenced, and used to analyze genomic DNA blots from A431 and normal cells. These data indicate that the aberrant RNA is created by a gene rearrangement within chromosome 7, resulting in a fusion of the 5' portion of the EGF receptor gene to an unidentified region of genomic DNA. The unidentified sequences are amplified to about the same degree (20- to 30-fold) as the EGF receptor sequences. In situ hybridization to chromosomes from normal cells and A431 cells show that both the EGF receptor gene and the unidentified DNA are localized to the p14-p12 region of chromosome 7. By using cDNA fragments to probe DNA blots from mouse-A431 somatic cell hybrids, the rearranged receptor gene was shown to be associated with translocation chromosome M4.     

Characterization of a cDNA clone for a rare mRNA modulated by ovariectomy in mammary carcinomas

A complementary DNA (cDNA) clone (p13) for a rare mRNA was isolated from a cDNA library generated from total polyA+ RNA of 14-day lactating rat mammary gland. In vitro translation of the positively selected mRNA from p13 cDNA revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) a polypeptide of 24 kDa. The p13 cDNA clone hybridized on northern blots predominantly to approximately 1100 base size RNA and weakly to approximately 3800 base size RNA from lactating mammary gland. It hybridized only to approximately 3800 base size RNA from rat liver. Southern blot analysis of genomic DNA showed differences in gene organization in mammary gland and liver. The mRNA level for the 24 kDa polypeptide was higher in 7-12 DMBA-induced tumor and lower in the MTW9 carcinoma as compared to lactating mammary gland. After ovariectomy, the mRNA level in mid pregnant gland increased but was reduced in the 7-12 DMBA tumors.     

The genomic organization of the rat AT1 angiotensin receptor.

The AT1 receptor subtype modulates all of the hemodynamic effects of the vasoactive peptide, angiotensin II. In this report, we investigate the genomic organization of this important receptor. A rat genomic library was screened with fragments from the 5' region of a previously cloned cDNA, pCa18b, encoding the rat AT1 receptor. Two lambda clones were isolated and the hybridizing restriction fragments were sequenced. Comparison of the genomic and cDNA sequences reveals that the rat AT1 receptor has three exons. Two of the exons encode 5' untranslated sequence while the third exon encompasses the entire coding region, a small portion of the 5' untranslated region and the entire 3' untranslated sequence. Further analysis of the genomic sequence 5' to the start site of pCa18b demonstrates typical sequence motifs found in many eukaryotic promoters including a TATA box, a cap site and a potential Sp1 binding site. Southern analysis of genomic DNA indicates that the AT1 receptor subtype represented by pCa18b is encoded by one gene within the rat genome.