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A R Shuldiner

Publications and source records attributed to A R Shuldiner.

88 records · Page 5Linked to original sources
PubMed1991

Two nonallelic insulin genes in Xenopus laevis are expressed differentially during neurulation in prepancreatic embryos.

Insulin, traditionally regarded as a metabolic hormone, also can potently stimulate growth and differentiation in many cell types. To study further the potential role of insulin during early embryogenesis, we have used the amphibian Xenopus laevis, a versatile model of vertebrate development. Using (i) nucleotide sequences of two previously cloned cDNAs that correspond to two different nonallelic Xenopus insulin genes (both of which are expressed in the adult pancreas) and (ii) a modification of the highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) method developed in our laboratory, designated RNA template-specific PCR (RS-PCR), we now find that mRNAs for both Xenopus insulins I and II are present in mature (stage VI) oocytes but not in less-mature oocytes (stages I and IV) or in unfertilized eggs. The Xenopus insulin II gene is differentially expressed during early neurulation (stage 13), while only the insulin I gene is expressed at stage 21, when the neural tube is closing and cephalization is beginning. During later stages (i.e., stage 26) there is a region in the head that appears to be transcribing only the insulin I gene, while mRNAs for both insulins I and II are present in the body region. These findings show that the two nonallelic insulin genes are expressed differentially in Xenopus embryos in a stage- and region-specific manner; because appropriate receptors are also present, we suggest a role for insulin during early nervous system development well before the emergence of pancreatic beta cells.

Alleles↗
PubMed1991

Genes encoding receptors for insulin and insulin-like growth factor I are expressed in Xenopus oocytes and embryos.

Insulin and insulin-like growth factor I (IGF-I) initiate their metabolic, growth, and differentiation effects through binding to the insulin receptor and the IGF-I receptor, two members of the tyrosine kinase family of receptors. To study the role of these peptides and receptors in early development, we used the polymerase chain reaction and embryo-derived RNA to generate partial cDNA sequences of the insulin receptor and IGF-I receptor from the amphibian Xenopus laevis. Three unique tyrosine kinase-related sequences were obtained. Two of the nucleotide sequences, XTK 1a and XTK 1b, corresponded to peptide that share 92% amino acid identity, and each is 89% identical to the human insulin receptor. The third sequence, XTK 2, corresponds to a peptide that has 92% amino acid identity with the human IGF-I receptor but only 80% identity with XTK 1a and XTK 1b. On the basis of these similarities, the pattern of conserved amino acids, and the tetraploid nature of the Xenopus genome, we suggest that XTK 1a and XTK 1b most likely represent the product of two different nonallelic insulin receptor genes, while XTK 2 may be one of the probable two Xenopus IGF-I receptor genes. By reverse transcription-polymerase chain reaction and gene-specific hybridization, expression of the three XTK sequences was detected in the oocyte, unfertilized egg, and embryos through gastrulation, neurulation, and tailbud stages. Competition binding assays with Xenopus membrane preparations demonstrated insulin receptors and IGF-I receptors in older tadpoles. IGF-I receptors were also present in oocytes, eggs, and gastrula embryos. By contrast, insulin binding was present but atypical in oocytes and was barely detected in eggs and gastrula embryos. The expression of receptors for insulin and IGF-I in early Xenopus embryos and their apparent distinct developmental regulation suggest that these molecules and their ligands may be important in early Xenopus development.

Amino Acid Sequence↗
PubMed1991

Ligase-free subcloning: a versatile method to subclone polymerase chain reaction (PCR) products in a single day.

Often, it is convenient to subclone polymerase chain reaction (PCR) products into a plasmid vector for subsequent replication in bacteria, but conventional subcloning methods often fail. We report a rapid and versatile method to subclone PCR products directionally into a specific site of virtually any plasmid vector. The procedure requires only four primers, does not require DNA ligase, and may be accomplished in a single day. Ligase-free subcloning is performed by incorporating into the PCR primers sequences at the 5' ends that result in PCR products whose 3' ends are complementary to the 3' ends of the recipient linearized plasmid. The PCR product and the linearized plasmid are spliced together in a second PCR reaction in which Taq polymerase extends the complementary overlapping 3' ends (ligation by overlap extension). Denaturation followed by heterologous reannealing and cyclization results in a cyclic recombinant plasmid with two nicks that may be used directly to transform competent Escherichia coli. In our hands, ligase-free subcloning is rapid, and offers many advantages over existing strategies.

Bacteriophage lambda↗
PubMed1991

RNA template-specific PCR: an improved method that dramatically reduces false positives in RT-PCR.

We report a novel modification of the reverse transcription PCR method, designated RNA template-specific PCR. With this approach, the 5' end of the first strand is tagged with a unique nucleotide sequence during reverse transcription that may then be exploited to amplify preferentially RNA-derived sequences. In our hands, RNA template-specific PCR retains the sensitivity of the traditional method, but greatly reduces the frequency of false positives, and virtually eliminates carryover contamination from PCR products amplified in previous experiments.

Animals↗
PubMed1990

RNA template-specific polymerase chain reaction (RS-PCR): a novel strategy to reduce dramatically false positives.

We report a novel modification of the reverse transcription-polymerase chain reaction method that we have dubbed RNA template-specific PCR (RS-PCR). With this approach, the 5' end of the first strand is tagged with a unique nucleotide sequence during reverse transcription which may then be exploited to amplify preferentially RNA-derived sequences. In our hands, RS-PCR greatly reduces the frequency of false positives and virtually eliminates carryover contamination from DNA fragments amplified in previous experiments.

Animals↗
PubMed1990

Evidence that Xenopus laevis contains two different nonallelic insulin-like growth factor-I genes.

Using the polymerase chain reaction (PCR), we have amplified and characterized partial nucleotide sequences of two distinct insulin-like growth factor-I genes (designated IGF-I' and IGF-I") from the amphibian, Xenopus laevis. The amplified fragments encoded much of the coding region of the mature peptide (exon III in mammalian IGF-I genes), and exhibited 93% similarity to each other, and 68-82% similarity to mammalian IGF-I amino acid sequences. Southern blot analysis using genomic DNA from a homozygous frog revealed that these two genes are nonallelic in a single organism, like the two nonallelic genes encoding Xenopus insulins that we have characterized previously. Furthermore, both IGF-I mRNAs are expressed in similar quantities in adult liver.

Alleles↗
PubMed1990

The insulin-like growth factor I (IGF-I) gene is expressed in chick embryos during early organogenesis.

A definition of the role of IGF-I in differentiation and development requires a detailed understanding of its expression and tissue-specific regulation in embryogenesis. Standard techniques for analysis of IGF-I gene expression are not sufficiently sensitive for studies in early embryos. We have used the highly sensitive polymerase chain reaction (PCR) to study IGF-I gene expression in whole chick embryos from the late blastula stage (E0 = laying) through the end of organogenesis (day 8), and in liver, brain and pancreas during mid-late embryogenesis and perinatally (hatching = day 21). Although at low levels in the blastoderm and gastrula, IGF-I mRNA was detectable in the whole embryo in all stages studied, with a tendency of the signal to increase with age during the first week of embryogenesis. In mid- and late embryogenesis, we easily detected IGF-I mRNA transcripts in pancreas and brain while the levels in the liver were barely detectable. Liver IGF-I mRNA increased markedly at the peak of postnatal growth (day 50). These studies suggest that while the major source of postnatal IGF-I may be the liver, extrahepatic tissues may be the predominant source of IGF-I during prenatal chicken development.

Animals↗
PubMed1989

Xenopus laevis contains two nonallelic preproinsulin genes. cDNA cloning and evolutionary perspective.

We undertook the cloning of preproinsulin cDNAs from the South African clawed toad, Xenopus laevis, in order to study the role of insulin during embryogenesis in this species. We found that X. laevis contains two different preproinsulin cDNAs, both of which code for peptides containing 106 amino acids of typical structure but which differ by eight amino acids: one in the signal peptide, two in the B-chain, four in the C-peptide, and one in the A-chain. Southern blot analysis indicates that the two preproinsulin cDNAs identified correspond to two different nonallelic genes which we believe arose through a recent gene duplication within the amphibian radiation possibly during the development of tetraploidy in this species. Both genes are expressed, since we have recently identified the two corresponding insulins in pancreatic extracts of adult toads (Shuldiner, A.R., Bennett, C., Robinson, E.A., and Roth, J. (1989) Endocrinology, in press). These cDNAs represent the first amphibian preproinsulin sequences to be elucidated.

Alleles↗
PubMed1989

Isolation and characterization of two different insulins from an amphibian, Xenopus laevis.

The South African clawed toad, Xenopus laevis, is a versatile laboratory model of vertebrate development. To study the role of insulin during embryogenesis, we have recently cloned preproinsulin cDNAs from this species. Unexpectedly, we identified two preproinsulin cDNAs corresponding to two different nonallelic genes that code for similar but distinctly different insulins. We now report the isolation, amino acid sequence, and characterization of both of these insulins from pancreatic extracts of adult toads, confirming that both Xenopus preproinsulin genes are expressed. Xenopus insulins represent the first amphibian insulins to be characterized. Xenopus insulin I and Xenopus insulin II are more similar to each other than they are to insulins of other species. In addition, Xenopus insulins are more similar to mammalian and bird insulins, than they are to fish insulins, implying a closer evolutionary link to terrestrial vertebrates than to most aquatic vertebrates. A homogeneous preparation of Xenopus insulin I showed high reactivity in a pork insulin RIA. Xenopus insulin I was approximately 2-fold more potent than pork insulin in binding to insulin receptors on human IM-9 lymphocytes and 1.5-fold more potent than pork insulin in stimulating glucose oxidation in rat adipocytes. We were unable to purify Xenopus insulin II sufficiently for immunological and biological characterization.

Amino Acid Sequence↗
PubMed1985

N-cyclo-[Leu5]enkephalin: a rational approach for the synthesis of conformationally restricted cyclic pentapeptides.

The enkephalins are neuropeptides belonging to the class of endogenous opioids. The conformationally restricted analog, N-cyclo-[Leu5]enkephalin, was recently synthesized. Since the synthesis of cyclic pentapeptides which lack proline and contain amino acids with bulky side chains is problematic, the synthesis, purification, and analytical characterization of N-cyclo-[Leu5]enkephalin is described in detail. This conformationally restricted cyclic pentapeptide was prepared from H-Gly-Phe-Leu-[O-(2,6-dichlorobenzyl)-Tyr]-Gly-NHNH2, which was synthesized by the solid-phase method of peptide synthesis. Cyclization was accomplished through an azide intermediate at high dilution, using high-pressure liquid chromatography to monitor the reaction. The desired cyclic monomer was isolated and purified by semipreparative HPLC. The structure of the purified cyclic product was confirmed by multiple chemical techniques including amino acid analysis, lack of an amino terminus (as assessed by reaction with ninhydrin and Edman reagent), and mass spectroscopy.

Amino Acids↗
PubMedDate not supplied

Molecular scanning of the beta-3-adrenergic receptor gene in Pima Indians and Caucasians.

BACKGROUND: The beta-3-adrenergic receptor (beta3AR) stimulates lipolysis and thermogenesis in adipocytes. The Trp64Arg beta3AR variant is associated in some, but not all, studies with an earlier onset of Type 2 diabetes mellitus and features of the insulin resistance syndrome. Functional studies as to the role of the Trp64Arg variant have been inconclusive. Earlier studies screened the beta3AR gene in only ten obese, diabetic Pima Indians. Potentially another yet to be identified polymorphism in the beta3AR gene in linkage disequilibrium with the Trp64Arg polymorphism could explain the findings in the association and functional studies. METHODS: We scanned the beta3AR gene in 20 diabetic Pima subjects and 20 Caucasian subjects using single stranded conformational polymorphism (SSCP) analysis. Variants were sequenced using dideoxy sequence analysis and further characterized using allele specific oligonucleotide hybridization (ASO) and RNA template specific-polymerase chain reaction (RS-PCR) assays. RESULTS: We found a guanine to thymidine substitution in the first intron, 14 bases from the splice donor site in both groups. In virtually all subjects, only two haplotypes were detected, Trp64/g1856 and Arg64/t1856, indicating that the g1856t polymorphism is in linkage disequilibrium with the Trp64Arg polymorphism. The g1856t substitution introduces a new consensus splice donor site which, if used, would encode a truncated protein. RNA levels of the two beta3AR alleles were approximately equal in omental adipose tissue of heterozygotes. No aberrantly spliced beta3AR mRNA was detected, indicating that the new consensus splice donor site is not used in vivo. CONCLUSION: The g1856t polymorphism is in linkage disequilibrium with the Trp64Arg variant, but does not appear to have a functional role.

Amino Acid Substitution↗
PubMedDate not supplied

Molecular scanning for mutations in the insulin receptor substrate-1 (IRS-1) gene in Mexican Americans with Type 2 diabetes mellitus.

BACKGROUND: Insulin receptor substrate-1 (IRS-1) is an endogenous substrate for the insulin receptor tyrosine kinase, which plays an important role in insulin signaling. Mutations in the IRS-1 gene are associated in some populations with obesity and Type 2 diabetes. METHODS: To determine whether variation in the IRS-1 gene contributes to genetic susceptibility to insulin resistance and Type 2 diabetes in Mexican Americans, the entire coding region of the IRS-1 gene was screened for variation in 31 unrelated subjects with Type 2 diabetes using single-stranded conformational polymorphism analysis (SSCP) and dideoxy sequence analysis. Variants encoding amino acid substitutions were genotyped in 27 unrelated nondiabetic Mexican Americans and in all family members of subjects containing these variants, and association analyses were performed. To trace the ancestral origins of the variants, Iberian Caucasians and Pima Indians were also genotyped. RESULTS: Eight single base changes were found: four silent polymorphisms and four missense mutations (Ala94Thr, Ala512Pro, Ser892Gly and Gly971Arg). Allele frequencies were 0.009, 0.017, 0.017 and 0.043, respectively. There were no significant associations of any of these variants with diabetes, glucose or insulin levels during an oral glucose tolerance test, or with body mass index (BMI) in Mexican American families except for a modest association between the Ala94Thr variant and decreased BMI (30.4 kg/m(2) vs 24.0 kg/m(2); p=0.035). None of these four missense mutations were detected in Pima Indians. In Iberian Caucasians, neither Ala94Thr nor Ser892Gly were detected, and Ala512Pro was detected in only 0/60 diabetic patients and 1/60 nondiabetic controls. Gly971Arg was relatively more common in Iberian Caucasians with 12/58 diabetic patients and 7/60 nondiabetic controls being heterozygous for this variant (p=0.21 for comparison between diabetic and nondiabetic subjects). CONCLUSIONS: Ala94Thr, Ala512Pro and Ser892Gly mutation are rare in the populations studied. Gly971Arg, is more common in Mexican Americans and Caucasians, but is not a major contributor to genetic susceptibility to Type 2 diabetes.

Adult↗