Human prostatic acid phosphatase: cDNA cloning, gene mapping and protein sequence homology with lysosomal acid phosphatase

The cDNAs encoding human prostatic acid phosphatase were cloned and characterized. The mRNAs contain 3' noncoding regions of heterogeneous sizes 646, 1887 or 1913 nucleotides. A dimer and a monomer of the conserved Alu-repeats are present in the longer 3' noncoding sequences. The complete sequence of 354 amino acids for the mature enzyme was determined by sequencing both cDNA and protein. Human prostatic and lysosomal acid phosphatases exhibit 50% sequence homology, including five Cys residues and two putative N-linked glycosylation sites. The Acp-3 gene coding for human prostatic acid phosphatase was mapped onto chromosome 3 in this investigation. The Acp-2 gene coding for lysosomal acid phosphatase has previously been located on chromosome 11, while the Acp-1 gene coding for red blood cell acid phosphatase is on chromosome 2.     

cDNA cloning of human apoA-I: Amino acid sequence of preproapoA-I

ds-cDNA to human liver apoA-I mRNA has been cloned. Nucleic acid sequence analysis revealed that apoA-I mRNA codes for a precursor apolipoprotein, preproapoA-I, which contains 24 amino acids on the NH₂-terminal end of the mature plasma apoA-I. Eighteen amino acids are contained within the hydrophobic prepeptide (Met-Lys-Ala-Ala-Val-Leu-Thr-Leu-Ala-Val-Leu-Phe-Leu-Thr-Gly-Ser-Gln-Ala) followed by a 6 amino acid propeptide (Arg-His-Phe-Top-Gln-Gln). Our results on human apoA-I are in agreement with the partial sequence of the precursor of rat apoA-I with respect to the length of the precursor sequence, location of the prepeptide, and the presence of an unusual propeptide sequence terminating in a neutral dipeptide Gln-Gln. A detailed analysis of the primary structure of normal human apoA-I mRNA will be essential to our ultimate understanding of the processing of human apoA-I and diseases characterized by molecular defects in apoA-I structure and function.     

Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase

The cDNA encoding human ferrochelatase [EC 4.99.1.1] was isolated from a human placenta cDNA library in bacteriophage lambda gt11 by screening with a radiolabeled fragment of mouse ferrochelatase cDNA. The cDNA had an open reading frame of 1269 base pairs (bp) encoding a protein of 423 amino acid residues (Mr. 47,833) with alternative putative polyadenylation signals in the 3' non-coding regions and poly (A) tails. Amino acid sequencing showed that the mature protein consists of 369 amino acid residues (Mr. 42,158) with a putative leader sequence of 54 amino acid residues. The human enzyme showed an 88% identity to mouse enzyme and 46% to yeast enzyme. Northern blot analysis showed two mRNAs of about 2500 and 1600 bp for ferrochelatase in K562 and HepG2 cells. As full-length cDNA for human ferrochelatase is now available, molecular lesions related to erythropoietic protoporphyria can be characterized.     

The cloning and nucleotide sequence of human ST2L cDNA

The ST2 gene is a member of the IL-1 receptor family and is hypothesized to be involved in helper T cell function, but its functional ligand and physiological role remain unknown. We have cloned the human ST2L cDNA that encodes a distinct type of membrane-bound ST2 protein. The predicted 556-amino-acid sequence showed 67% identity to the mouse ST2L protein. The human ST2 gene (IL1RL1) contains 13 exons and spans 40 kb in length. Its exon-intron organization was elucidated from a registered human genomic sequence derived from chromosome 2q, which contains three other genes belonging to the IL-1 receptor family in an approximately 202-kb genomic region. The tissue distribution of ST2 expression was examined by RT-PCR, and the soluble form (ST2, IL1RL1-a) and ST2L (IL1RL1-b) appear to be expressed differentially. We also established stable transfectants of a human glioblastoma cell line, T98G, that express human ST2L constitutively, and we confirmed cell-surface expression of human ST2L protein on the transfectants.     

Molecular cloning and characterization of an interferon induced human cDNA with sequence homology to a mammalian peptide chain release factor.

Here we report the molecular cloning of several related human cDNAs from which a full-length sequence can be determined. The cDNAs encode a 2.8 kb mRNA that is strongly induced by interferon (IFN) gamma and the expression of which is not cell-restricted but observed in fibroblasts, macrophages and epithelial cells. The deduced amino acid sequence predicts a protein of 471 amino acids with high sequence similarity to a previously identified rabbit peptide chain release factor. Functional studies to demonstrate release factor activity showed that the protein encoded by this cDNA inhibited the readthrough activity of a yeast UGA suppressor tRNA in an in vitro translation system. The identification of this novel cDNA implies that translational control by IFN induced proteins may not be restricted to the initial steps of protein synthesis but may also act by regulation of peptide chain termination.     

Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues

Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions (Furuse, M., T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 123:1777-1788). To analyze and modulate the functions of tight junctions, it would be advantageous to know the mammalian homologues of occludin and their genes. Here we describe the nucleotide sequences of full length cDNAs encoding occludin of rat-kangaroo (potoroo), human, mouse, and dog. Rat-kangaroo occludin cDNA was prepared from RNA isolated from PtK2 cell culture, using a mAb against chicken occludin, whereas the others were amplified by polymerase chain reaction based on the sequence found around the human neuronal apoptosis inhibitory protein gene. The amino acid sequences of the three mammalian (human, murine, and canine) occludins were very closely related to each other (approximately 90% identity), whereas they diverged considerably from those of chicken and rat-kangaroo (approximately 50% identity). Implications of these data and novel experimental options in cell biological research are discussed.     

Molecular cloning, cDNA sequence, and bacterial expression of human glutamine:fructose-6-phosphate amidotransferase.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) has recently been shown to be an insulin-regulated enzyme that plays a key role in the induction of insulin resistance in cultured cells. As a first step in understanding the molecular regulation of this enzyme the human form of this enzyme has been cloned and the functional protein has been expressed in Escherichia coli. A 3.1-kilobase cDNA was isolated which contains the complete coding region of 681 amino acids. Expression of the cDNA in E. coli produced a protein of approximately 77 kDa and increased GFAT activity 4.5-fold over endogenous bacterial levels. Recombinant GFAT activity was inhibited 51% by UDP-GlcNAc whereas bacterial GFAT activity was insensitive to inhibition by UDP-GlcNAc. On the basis of these results we conclude that: 1) functional human GFAT protein was expressed, and 2) the cloned human cDNA encodes both the catalytic and regulatory domains of GFAT since the recombinant GFAT was sensitive to UDP-GlcNAc. Overall, the development of cloned GFAT molecular probes should provide new insights into the development of insulin resistance by allowing quantitation of GFAT mRNA levels in pathophysiological states such as non-insulin-dependent diabetes mellitus and obesity.     

cDNA cloning and sequence determination of pig gastric (H+ + K+)-ATPase

Complementary DNA to pig gastric mRNA encoding (H+ + K+)-ATPase was cloned, and its amino acid sequence was deduced from the nucleotide sequence. The enzyme contained 1034 amino acid residues (Mr. 114,285) including the initiation methionine. The sequence of pig (H+ + K+)-ATPase was highly homologous with that of the corresponding enzyme from rat, but had high degree of synonymous codon changes. Potential sites of phosphorylation by cAMP-dependent protein kinase and N-linked glycosylation sites were identified. The amino terminal region contained a lysine-rich sequence similar to that of the alpha subunit of (Na+ + K+)-ATPase, although a cluster of glycine residues was inserted into the sequence of the (H+ + K+)-ATPase. As the pig enzyme is advantageous for biochemical studies, the information of the primary structure is useful for further detailed studies.     

Molecular cloning and sequence analysis of cDNA encoding human prostatic acid phosphatase

lambda gt11 clones encoding human prostatic acid phosphatase (PAP) (EC 3.1.3.2) were isolated from human prostatic cDNA libraries by immunoscreening with polyclonal antisera. Sequence data obtained from several overlapping clones indicated that the composite cDNAs contained the complete coding region for PAP, which encodes a 354-residue protein with a calculated molecular mass of 41,126 Da. In the 5'-end, the cDNA codes for a signal peptide of 32 amino acids. Direct protein sequencing of the amino-terminus of the mature protein and its proteolytic fragments confirmed the identity of the predicted protein sequence. PAP has no apparent sequence homology to other known proteins. However, both the cDNA clones coding for human placental alkaline phosphatase and PAP have an alu-type repetitive sequence about 900 nucleotides downstream from the coding region in the 3'-untranslated region. Two of our cDNA clones differed from others at the 5'-ends. RNA blot analysis indicated mRNA of 3.3 kb. We are continuing to study whether acid phosphatases form a gene family as do alkaline phosphatases.     

Molecular cloning and nucleotide sequence of human alpha 1 acid glycoprotein cDNA

A cDNA clone has been isolated from a pEX expression library that encodes alpha 1 acid glycoprotein. We present the complete nucleotide sequence encoding this protein and compare the derived amino acid sequence with pre-existing data.     

Molecular cloning and nucleotide sequence of a human renin cDNA fragment.

We have studied human renin messenger RNA by hybridization with the mouse submaxillary gland (SMG) renin cDNA probe. The human kidney messenger RNA is about 1.6 kilobase (kb) long, similarly to the mouse SMG renin mRNA. A kidney renin cDNA clone of 1.1 kb length was obtained. A comparison of nucleotide sequences of mouse and human cDNA clones reveals conservation of residues involved in catalytic mechanisms and a potential glycosylation site. The human renin molecular probe allowed us to study renin expression in human chorionic tissue. The chorionic and kidney renin messenger RNAs are similar in length. The Southern blot analysis reveals the presence of a single renin gene in human DNA.     

Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor

Amino acid sequences of four peptide fragments of human hepatocyte growth factor purified from the plasma of patients with fulminant hepatic failure were determined. Based on the amino acid sequence of one of the fragments, two oligodeoxyribonucleotide mixtures were synthesized and used to screen a human placenta cDNA library. On the screening, two overlapping cDNA clones for human hepatocyte growth factor were isolated and the nucleotide sequence of the cDNA was determined. The entire primary structure of the protein was deduced from the sequence. The protein consists of 728 amino acid residues, including a possible signal peptide at the N-terminus. The sequence revealed that the heavy and light chains which comprise the protein are encoded by the same mRNA and are produced from a common translation product by proteolytic processing.     

cDNA cloning of pig testicular lactate dehydrogenase-C, thermal stability of the expressed enzyme, and polymorphism among strains

Pig testicular lactate dehydrogenase-C (LDHC) cDNA was cloned and sequenced. The deduced sequence of 332 amino acids from pig LDHC shows 73% and 67% identity with that of pig LDHA (muscle) and LDHB (heart) respectively, whereas pig LDHA and LDHB isozymes shows 74% sequence identity. Pig and mouse LDHC cDNAs were subcloned into bacterial expression vector, and the expressed pig LDHC isozyme was shown to be as thermally stable as mouse LDHC isozyme. Pig genomic DNAs from Chinese Meishan, English Yorkshire, Danish Landrace and American Duroc were shown to exhibit polymorphic sites for restriction enzymes EcoRI, BamHI and PstI.     

The rabbit muscle phosphofructokinase gene: cDNA cloning and sequencing

A partial cDNA for rabbit muscle phosphofructokinase (Rm-PFK) has been cloned and sequenced. The nucleotide sequence of the cDNA agrees with the previously determined Rm-PFK genomic sequence. In addition, the amino acid sequence deduced from the cDNA is nearly identical to the Rm-PFK sequence previously determined by peptide analysis. A significant degree of homology exists when the amino acid sequence of Rm-PFK is compared with the sequences of Bacillus stearothermophilus PFK or Escherichia coli PFK-1. The cloning and sequencing of the PFK cDNA fragment represents significant progress toward the long term goal of using site-specific mutagenesis to investigate the structure-function relationships in this allosteric enzyme.     

Giant panda ribosomal protein S14: cDNA, genomic sequence cloning, sequence analysis, and overexpression

RPS14 is a component of the 40S ribosomal subunit encoded by the RPS14 gene and is required for its maturation. The cDNA and the genomic sequence of RPS14 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology and touchdown-PCR, respectively; they were both sequenced and analyzed. The length of the cloned cDNA fragment was 492 bp; it contained an open-reading frame of 456 bp, encoding 151 amino acids. The length of the genomic sequence is 3421 bp; it contains four exons and three introns. Alignment analysis indicates that the nucleotide sequence shares a high degree of homology with those of Homo sapiens, Bos taurus, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus laevis, and Danio rerio (93.64, 83.37, 92.54, 91.89, 87.28, 84.21, and 84.87%, respectively). Comparison of the deduced amino acid sequences of the giant panda with those of these other species revealed that the RPS14 of giant panda is highly homologous with those of B. taurus, R. norvegicus and D. rerio (85.99, 99.34 and 99.34%, respectively), and is 100% identical with the others. This degree of conservation of RPS14 suggests evolutionary selection. Topology prediction shows that there are two N-glycosylation sites, three protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, four N-myristoylation sites, two amidation sites, and one ribosomal protein S11 signature in the RPS14 protein of the giant panda. The RPS14 gene can be readily expressed in Escherichia coli. When it was fused with the N-terminally His-tagged protein, it gave rise to accumulation of an expected 22-kDa polypeptide, in good agreement with the predicted molecular weight. The expression product obtained can be purified for studies of its function.