Ellson Y. Chen

Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand.

Structural features of v‐kit, the oncogene of HZ4 feline sarcoma virus, suggested that this gene arose by transduction and truncation of cellular sequences. Complementary DNA cloning of the human proto‐oncogene coding for a receptor tyrosine kinase confirmed this possibility: c‐kit encodes a transmembrane glycoprotein that is structurally related to the receptor for macrophage growth factor (CSF‐1) and the receptor for platelet‐derived growth factor. The c‐kit gene is widely expressed as a single, 5‐kb transcript, and it is localized to human chromosome 4 and to mouse chromosome 5. A c‐kit peptide antibody permitted the identification of a 145,000 dalton c‐kit gene product that is inserted in the cellular plasma membrane and is capable of self‐phosphorylation on tyrosine residues in both human glioblastoma cells and transfected mouse fibroblasts. Our results suggest that p145c‐kit functions as a cell surface receptor for an as yet unidentified ligand. Furthermore, carboxy‐ and amino‐terminal truncations that occurred during the viral transduction process are likely to have generated the transformation potential of v‐kit.

Overview of manual and automated DNA sequencing by the dideoxy chain termination method

Since the initial introduction of the dideoxy chain termination method, DNA sequencing has undergone rapid improvement, mainly in the areas of DNA polymerases, deoxynucleotide analogs, and polyacrylamide gel electrophoresis. In this article we review these improvements and their applications to manual DNA sequencing, and we present a brief evaluation of commercial efforts to automate DNA sample preparation, separation, and data acquisition. Included are instruments currently available as well as new concepts potentially amenable to automation.

Human transforming growth factor-α: Precursor structure and expression in E. coli

Abstract Transforming growth factor-α (TGF-α) is secreted by many human tumors and can induce the reversible transformation of nontransformed cell lines. Using long synthetic deoxyoligonucleotides as hybridization probes we isolated an exon coding for a portion of TGF-α from a human genomic DNA library. Utilizing this exon as a probe, a cell line derived from a human renal cell carcinoma was identified as a source of TGF-α mRNA. A cloned TGF-α cDNA was isolated from a cDNA library prepared using RNA from this cell line, and was found to encode a precursor polypeptide of 160 amino acids. The 50 amino acid mature TGF-α produced by expression of the appropriate coding sequence in E. coli binds to the epidermal growth factor receptor and induces the anchorage independence of normal mammalian cells in culture.

Mutations in GPC3, a glypican gene, cause the Simpson-Golabi-Behmel overgrowth syndrome

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition characterized by pre-and postnatal overgrowth with visceral and skeletal anomalies. To identify the causative gene, breakpoints in two female patients with X;autosome translocations were identified. The breakpoints occur near the 5′, and 3′, ends of a gene, GPC3, that spans more than 500 kilobases in Xq26; in three families, different microdeletions encompassing exons cosegregate with SGBS. GPC3 encodes a putative extracellular proteoglycan, glypican 3, that is inferred to play an important role in growth control in embryonic mesodermal tissues in which it is selectively expressed. Initial western- and ligand-blotting experiments suggest that glypican 3 forms a complex with insulin-like growth factor 2 (IGF2), and might thereby modulate IGF2 action.

X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein.

Ectodermal dysplasias comprise over 150 syndromes of unknown pathogenesis. X–linked anhidrotic ectodermal dysplasia (EDA) is characterized by abnormal hair, teeth and sweat glands. We now describe the positional cloning of the gene mutated in EDA. Two exons, separated by a 200–kilobase intron, encode a predicted 135–residue transmembrane protein. The gene is disrupted in six patients with X;autosome translocations or submicroscopic deletions; nine patients had point mutations. The gene is expressed in keratinocytes, hair follicles, and sweat glands, and in other adult and fetal tissues. The predicted EDA protein may belong to a novel class with a role in epithelial–mesenchymal signalling.

The human growth hormone locus: Nucleotide sequence, biology, and evolution ☆

The human chromosomal growth hormone locus contained on cloned DNA and spanning approximately 66,500 bp was sequenced in its entirety to provide a framework for the analysis of its biology and evolution. This locus evolved by a series of duplications and contains in its present form five genes which display a remarkably high degree of sequence identity (approximately 95%) in all their domains. The DNA sequence of the locus reveals the presence of 48 middle repetitive sequence elements of the Alu type and one member of the KpnI family, all located in the intergenic regions. The expression of each gene was examined by screening pituitary and placental cDNA libraries by using gene-specific oligonucleotides. According to this analysis, the hGH-N gene is transcribed exclusively in the pituitary, whereas the other four genes (hCS-L, hCS-A, hGH-V, hCS-B) are expressed only in placental tissue, at levels characteristic for each gene. Particular DNA sequences found upstream of the individual promoter regions might account for the observed tissue specificity and different transcriptional activity of the genes. The hCS-L gene carries a G to A transition in a sequence used by the other four genes as an intronic 5 splice donor site. This mutation results in a different splicing pattern and, hence, in a novel sequence of the hCS-L gene mRNA and the deduced polypeptide.

Primary structure and mRNA localization of protein F1, a growth-related protein kinase C substrate associated with synaptic plasticity.

Protein F1 is a neuron‐specific, synaptic‐enriched, membrane‐bound substrate of protein kinase C (PKC) whose phosphorylation is related to synaptic plasticity in the adult. The sequence of 26 N‐terminal amino acids was determined from purified rat protein F1. A 78‐mer synthetic oligonucleotide designed from the partial N‐terminal sequence enabled identification of protein F1 cDNA clones in a rat brain library. F1 protein is a 226 amino acid protein encoded by a 1.5 kb brain‐specific, developmentally‐regulated mRNA. Transcripts for protein F1 can be detected at birth, and their level declines after maturation. A full‐length cDNA clone was transcribed and translated in vitro. Translation products could be immunoprecipitated with anti‐F1 antibodies. In situ hybridization analysis revealed protein F1 transcripts in hippocampal pyramidal cells, but not in granule cells. In cerebellum, granule cells contained protein F1 mRNA, while Purkinje cells did not. Co‐localization of protein F1 with protein kinase C‐II [PKC‐II (beta)], rather than PKC‐I (gamma) suggests that PKC‐II may phosphorylate protein F1.

Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli

The nucleotide sequence of the alkaline phosphatase (APase) gene (phoA) of Escherichia coli strain 294 has been determined. Pre-APase has a total of 471 amino acids (aa) including a signal sequence of 21 aa. The derived aa sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at positions 16 and 36, and glutamic acid instead of glutamine at position 197. Two open reading frames (ORF1 and ORF2) located downstream from phoA or upstream from proC have been found. ORF1 encodes a putative presecretory protein of 106 aa with a signal sequence of 21 or 22 aa. If this protein is actually produced, it may be one of the smallest periplasmic proteins in E. coli.

Sequence of human glucose-6-phosphate dehydrogenase cloned in plasmids and a yeast artificial chromosome

The sequence of 20,114 bp of DNA including the human glucose-6-phosphate dehydrogenase (G6PD) gene was determined. The region included a prominent CpG island, starting about 680 nucleotides upstream of the transcription start site, extending about 1050 nucleotides downstream of the start site, and ending just at the start of the first intron. The transcribed region from the start site to the poly(A) addition site covers 15,860 bp. The sequence of the 13 exons agreed with published cDNA sequence and for the 11 exons tested, with the corresponding sequence in a yeast artificial chromosome (YAC). The latter confirms YAC cloning fidelity at the DNA sequence level. Sixteen Alu sequences constitute 24% of the total sequence tract. Four were outside the borders of the mRNA transcript of the gene; all the others were found in a large (9858 bp) intron between exons 2 and 3. Two Alu clusters each contain Alus lying between the monomers of another.

Sequence analysis, cellular localization, and expression of a neuroretina adhesion and cell survival molecule

A cDNA for purpurin, a secreted 20,000 dalton neural retina cell adhesion and survival protein, has been sequenced and expressed in mammalian cells. Purpurin mRNA is found in both embryonic and adult retina, but not the brain, heart, or liver. The protein is highly concentrated in the neural retina between the pigmented epithelium and the outer segments of the photoreceptor cells; it is synthesized by photoreceptor cells. The predicted purpurin sequence contains 196 residues, has approximately 50% sequence homology with serum retinol binding protein, and is a member of the alpha-2 mu-globulin superfamily. Purpurin binds retinol and may play a major role in retinol transport across the interphotoreceptor cell matrix.