The structurally distinct form of pp60c-src detected in neuronal cells is encoded by a unique c-src mRNA.

A cellular src (c-src) cDNA clone was isolated from a chicken embryonic brain cDNA library and characterized by DNA sequence analysis. Comparison with the published sequence of a chicken genomic c-src clone indicated that the brain cDNA clone contained an 18-base-pair insertion located between exons 3 and 4 of the c-src gene. The six amino acids encoded by the insertion caused an alteration in the electrophoretic mobility of the c-src gene product similar to that of the structurally distinct form of the src protein detected in neuronal cultures.     

Molecular cloning and sequencing of a cDNA encoding a human alpha 1A adrenergic receptor.

DNA from a rat hippocampus cDNA library and sets of highly degenerate oligonucleotide primers directed toward conserved regions of previously cloned G-protein receptors were used in the polymerase chain reaction to selectively amplify and clone new members of this gene family. A human hippocampus cDNA library was screened with a 610 base pair fragment generated by PCR and a cDNA clone, H318/3, was isolated. The deduced amino acid sequence of this clone encoded a protein of 501 amino acids that showed strong sequence homology to previously cloned G-protein receptors. Nucleotide sequence analysis revealed clone H318/3 was 78% homologous to a rat alpha 1A adrenergic receptor with homology being 95% when comparisons were made in the region that lies between the first to the seventh transmembrane domains. Based on this high degree of sequence homology, we conclude that clone H318/3 represents a cDNA for a human alpha 1A adrenergic receptor.     

Isolation and characterization of related cDNA clones encoding skeletal muscle beta-tropomyosin and a low-molecular-weight nonmuscle tropomyosin isoform.

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.     

cDNA cloning and chromosomal localization (4q11-13) of a gene for statherin, a regulator of calcium in saliva.

On the basis of the known amino acid sequence of statherin, a human salivary protein, mixed synthetic oligonucleotides were synthesized and used to screen a cDNA library constructed from human parotid-gland mRNA. A cDNA clone coding for statherin was isolated from this library and has been completely sequenced. The cDNA represents a full-length (or nearly full-length) copy of an approximately 640-bp statherin mRNA. Statherin appears to be coded by a single-copy gene that maps to chromosome 4q11-4q13 when somatic-cell hybrids are used.     

A Novel Mesoderm-Specific cDNA Isolated from a Mouse Embryonal Carcinoma Cell Line

A novel mesoderm-specific cDNA clone has been isolated by differential screening of cDNA library from an embryonal carcinoma (EC) cell line MC12. The cDNA clone 121a is about 2.5 kb in length and apparently encodes a putative polypeptide of 335 amino acids which may be secreted or membrane anchored glycoprotein since it has a possible signal sequence and a potential N-linked glycosylation site. In situ hybridization using mouse embryos revealed that 121a expression was confined to mesoderm and its derivatives such as allantois, the mesodermal layer of amnion, chorion and yolk sac, somites, heart, etc. These findings suggest that 121 a may be essential for mesodermal differentiation or function, although nothing definite is known. Conservation of 121a homolog in mammals and even in Drosophila seems to support this presumption. Fluorescence in situ hybridization successfully localized 121a to B1 band of mouse chromosome 6.     

Cloning of a human liver microsomal UDP-glucuronosyltransferase cDNA.

A cDNA clone (HLUG 25) encoding the complete sequence of a human liver UDP-glucuronosyltransferase was isolated from a lambda gt11 human liver cDNA library. The library was screened by hybridization to a partial-length human UDP-glucuronosyltransferase cDNA (pHUDPGT1) identified from a human liver pEX cDNA expression library by using anti-UDP-glucuronosyltransferase antibodies. The authenticity of the cDNA clone was confirmed by hybrid-select translation and extensive sequence homology to rat liver UDP-glucuronosyltransferase cDNAs. The sequence of HLUG 25 cDNA was determined to be 2104 base-pairs long, including a poly(A) tail, and contains a long open reading frame. The possible site of translation initiation of this sequence is discussed with reference to a rat UDP-glucuronosyltransferase cDNA clone (RLUG 38).     

Binding of Murine Leukemia Virus Gag Polyproteins to KIF4, a Microtubule-Based Motor Protein

A cDNA clone encoding a cellular protein that interacts with murine leukemia virus (MuLV) Gag proteins was isolated from a T-cell lymphoma library. The sequence of the clone is identical to the C terminus of a cellular protein, KIF4, a microtubule-associated motor protein that belongs to the kinesin superfamily. KIF4-MuLV Gag associations have been detected in vitro and in vivo in mammalian cells. We suggest that KIF4 could be involved in Gag polyprotein translocation from the cytoplasm to the cell membrane.     

Isolation of a cDNA encoding the human GM2 activator protein

The GM2 activator protein is a glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. A human fibroblast cDNA library was screened with mixtures of oligonucleotide probes corresponding to four different areas of the amino acid sequence. A putative clone (821 bp) which gave positive signals to all four probe mixtures was purified and sequenced. The sequence was colinear with the sequence of 160 amino acids of the mature GM2 activator protein. Availability of the cDNA clone should facilitate investigation into function of the GM2 activator protein and also into genetic abnormalities underlying GM2 gangliosidosis AB variant.     

Molecular cloning and functional expression of cDNA encoding a cysteine proteinase inhibitor,cystatin, from Job's tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

A lambdaZAP II cDNA library was constructed from mRNA in immature seeds of the grass Job's tears. A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library. The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues. The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn. The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found. A remarkable characteristic of the peptide sequence of Job's-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice. The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The purified recombinant protein inhibited papain.     

cDNA sequence of human class III alcohol dehydrogenase

A human placental cDNA library was screened using oligonucleotide probes based on the peptide sequence of the human class III alcohol dehydrogenase. An incomplete cDNA clone covering most of the coding sequence of class III alcohol dehydrogenase was isolated from a human placental cDNA library. This was subsequently used as a probe to obtain a full-length clone from a human testicular library. The cDNA sequence codes for a protein that is identical to the enzyme purified from human liver. Southern analysis of human genomic DNA suggests that it may contain more than a single copy per haploid genome.     

Isolation and characterisation of a cDNA clone for a chlorophyll synthesis enzyme from Euglena gracilis. The chloroplast enzyme hydroxymethylbilane synthase (porphobilinogen deaminase) is synthesised with a very long transit peptide in Euglena

A cDNA expression library was constructed from light-grown Euglena gracilis poly(A)-rich RNA in lambda gt11. Antibodies to Euglena hydroxymethylbilane synthase, the third enzyme in the porphyrin biosynthetic pathway, were used to screen the library and a clone encoding part of the sequence of hydroxymethylbilane synthase was identified. This was used to rescreen the library and a full-length clone was isolated, which encoded not only the entire mature protein (Mr 36,927), but also an N-terminal extension of 139 amino acids. The deduced Mr of the whole polypeptide is 51,744, which corresponds to the size of the protein immunoprecipitated from the translation products of Euglena poly(A)-rich RNA. The mature protein is 60-70% similar to hydroxymethylbilane synthase from human erythrocytes and Escherichia coli. The sequence of the N-terminal extension has similarities to both the transit peptides of chloroplast proteins and those for the endoplasmic reticulum. This is the first report both of a cDNA clone for an enzyme of the chlorophyll biosynthetic pathway and of a putative transit peptide for a nuclear-encoded Euglena protein.     

Cell-free translation of silkmoth chorion mRNAs: Identification of protein precursors and characterization of cloned DNAs by hybrid-selected translation

The secretory silkmoth chorion proteins are synthesized as precursors bearing signal peptides. Precursors are detected upon cell-free translation of chorion mRNAs in the wheat germ system; they are processed into products identical in size to authentic chorion proteins when translation is performed in the presence of microsomal membranes from dog pancreas. Precursors corresponding to specific protein size classes and subclasses are identified by three approaches: comparison of precursors and products encoded by stage-specific mRNAs, comparison of precursors and products encoded by mRNAs specifically hybridizing to individual chorion cDNA clones, and comparison of relative amino acid compositions of precursors and authentic chorion proteins. Translation of stage-specific mRNA preparations indicates that, in general, the developmental changes of in vivo chorion protein synthesis are based on changes in concentrations of the corresponding mRNAs. Characterization of the precursors makes it possible to identify, for any chorion DNA clone, the protein subclass, a member of which is encoded by the clone sequence.     

Isolation and characterization of a cDNA clone encoding human aromatic L-amino acid decarboxylase

The nucleotide sequence of a cDNA clone that includes the entire coding region of human aromatic L-amino acid decarboxylase gene is presented. A human pheochromocytoma cDNA library was screened using an oligonucleotide probe which corresponded to a partial amino acid sequence of the enzyme purified from the human pheochromocytoma. The isolated cDNA clone encoded a protein of 480 amino acids with a calculated molecular mass of 53.9 kDa. The amino acid sequence Asn-Phe-Asn-Pro-His-Lys-Trp around a possible cofactor (pyridoxal phosphate) binding site is identical in human, Drosophila, and pig enzymes.     

Evolution of two major chorion multigene families as inferred from cloned cDNA and protein sequences

Complete or partial sequences are reported from six chorion cDNA clones of the silkmoth Antheraea polyphemus. The proteins encoded belong to the two major chorion protein classes, A and B, each of which is encoded by a multigene family. The sequence comparisons define some major features of the families and suggest how these genes may be evolving. Deletions and insertions might be involved in expanding or contracting internally repetitive regions. Sequence divergence is localized, thus defining sequence domains of distinct evolutionary properties and presumably distinct functions.     

Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene.

Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the alpha, beta, and intercalary heterochromatic regions. Screening of a lambda gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a lambda gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.     

Characterization of a cDNA coding for sex steroid-binding protein of human plasma

A cDNA (912 nucleotides) coding for human plasma sex steroid-binding protein (SBP) was characterized from a phage clone previously isolated by screening a Charon 21A human liver cDNA library with rat androgen binding protein (ABP) cDNA. The deduced amino acid sequence from the cDNA indicated that the insert was a partial clone coding for 281 amino acids starting with residue 92 (glycine) encompassing the alternating leucyl residues and the carboxyl-end 373 (histidine) as previously reported [(1986) Biochemistry 25, 7584]. The potential polyadenylation signal sequence ATTAAA is present as part of the 3'-coding region and the stop codon TAA. Both are followed by a short 20 untranslated nucleotides and a poly(A) tract of 49 nucleotides. Significant homologous sequences (76%) at the DNA level exist between human SBP and rat ABP which might suggest the possibility that both evolved from a common primordial gene. Demonstration of the presence of an SBP cDNA in a human liver cDNA library provides the first evidence that liver is the site of SBP biosynthesis.