Molecular cloning and sequence analysis of cDNA containing the entire coding region for human fetal liver cytochrome P-450

From a human fetal liver cDNA library, a cDNA clone (lambda HFL33) containing the entire coding region for a form of cytochrome P-450 related to P-450 HFLa was obtained. The clone was 1,971 bp long and had an open reading frame of 1,509 nucleotides coding for a 503 amino acid polypeptide. The nucleotide and the deduced amino acid sequences of lambda HFL33 were very similar to but clearly distinct from those of NF25 and HLp cDNAs, which code for forms of cytochrome P-450 in adult human liver. The deduced N-terminal amino acid sequence of the HFL33 protein was identical to that of P-450 HFLa.     

Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells

One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.     

Liver (B-type) phosphofructokinase mRNA. Cloning, structure, and expression

Mouse liver mRNA enriched in sequences coding for liver phosphofructokinase by polysome immunoadsorption was used as a template for the synthesis of cDNA. The double-stranded cDNA was inserted into the expression vector lambda gt11 and cloned. Preliminary identification of clones containing cDNA sequences for phosphofructokinase was made by screening the library with anti-rat liver phosphofructokinase serum and horseradish peroxidase-conjugated goat anti-rabbit IgG as second antibody. Subsequently, by selecting antibodies specific to fusion proteins expressed by putative clones and by reacting with Western blots of mouse liver proteins several clones were positively identified as containing liver phosphofructokinase sequences. A cDNA clone corresponding to 2708 nucleotides of liver phosphofructokinase mRNA was further characterized and sequenced. The liver phosphofructokinase mRNA has an open reading frame of 2343 nucleotides followed by a 3'-untranslated region of 303 nucleotides. The G/C-rich (76%) portion of the 5'-untranslated region precedes a characteristic translational start site of CCGCC(AUG). The mRNA coding sequence indicates that the liver phosphofructokinase subunit is composed of 780 amino acid residues and has a Mr of 85,000. Comparison of the deduced amino acid sequence of mouse liver phosphofructokinase with the known rabbit muscle phosphofructokinase shows 68% homology. The N-half of the liver phosphofructokinase has conserved substrate binding sites for ATP and fructose-6-P. The 25 C-terminal residues, which contain the ATP inhibitory site, are the least homologous (20%) but contain a putative phosphorylation site (Arg-Arg-X-X-Ser). The liver phosphofructokinase mRNA is under nutritional and hormonal regulation. The liver phosphofructokinase mRNA level increased 4-fold when previously starved mice were refed a high carbohydrate, fat-free diet. This increase in mRNA level was blocked by 50% by the administration of dibutyryl cAMP. The induction of liver phosphofructokinase mRNA by fasting/refeeding was also diminished in streptozotocin diabetic mice.     

Thymosin-beta 4 gene. Preliminary characterization and expression in tissues, thymic cells, and lymphocytes

A cDNA for rat thymosin-beta 4 was used to investigate the expression of this gene in different tissues, thymic cells, and lymphocytes. Hybridization analysis of total RNA from 13 rat tissues demonstrated the presence of an 800 nucleotides-long mRNA in all the tissues surveyed, with the highest levels in spleen, thymus, and lung. Examination of thymic cells showed that the thymosin-beta 4 gene is predominantly expressed in thymocytes. The thymosin-beta 4 mRNA was also studied in Ig+ and Ig- lymphocytes, being fourfold more abundant in Ig- than Ig+ splenic lymphocytes, whereas similar levels were found in both types of blood cells. The analysis of RNA from T cells at different maturation stages evidenced slight differences in their thymosin-beta 4 mRNA content, indicating that thymosin-beta 4 gene expression is not clearly related to the differentiation process of T cells. All these results do not support the roles for thymosin-beta 4 in cellular immunity and differentiation of lymphoid cells, suggesting a more general function for this peptide. Preliminary characterization of the human beta 4 gene by restriction analysis disclosed a complicated pattern consistent with multiple genes and/or introns. The analysis of genomic DNA from different species ranging from humans to Escherichia coli showed that this gene is only highly conserved in mammals.     

Molecular cloning and characterization of N-syndecan, a novel transmembrane heparan sulfate proteoglycan

A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain.     

The nucleotide sequence of a complete chicken delta-crystallin cDNA

The nucleotide sequence of a full length cDNA of delta-crystallin mRNA from chicken lens has been determined using a delta-crystallin cDNA clone (pB delta 11), which represents the mRNA sequence of 1530 nucleotides from the poly(A) junction but does not contain the 5'-terminal sequence of 44 nucleotides of the mRNA. The 5'-terminal sequence of the mRNA, absent in the cDNA clone, has been determined with a stretch of cDNA sequence by the primer extension procedure. The amino acid sequence deduced from the nucleotide sequence is consistent with the amino acid sequences of several tryptic peptides, the total amino acid composition, and the mol. wt. of delta-crystallin estimated by SDS-polyacrylamide gel electrophoresis. The computer-assisted analysis predicts high alpha-helical content throughout the polypeptide. Sequence analyses have revealed that gene 1 encodes the mRNA from which the cDNA clone was derived.     

Molecular cloning, cDNA structure and deduced amino acid sequence for the hormone-induced regulatory subunit (RII beta) of cAMP-dependent protein kinase from rat ovarian granulosa cells

The regulatory subunit of cAMP-dependent protein kinase designated RII beta (RII51) has previously been shown to be the product of a separate gene. This was accomplished by the molecular cloning of a partial cDNA clone estimated to lack 30-45 nucleotides of the 5' end of the coding region. We hereby report the isolation of a cDNA clone for RII beta from rat granulosa cells, extending 43 nucleotides further 5' compared with the previously published cDNA sequence, and from which the entire amino acid sequence (415 residues) of the rat RII beta protein can be deduced. A cAMP regulated mRNA of 3.2 kilobases (kb) for RII beta was detected by the isolated cDNA in rat Sertoli cells.     

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.     

Isolation and expression of cDNA for different forms of hepatocyte growth factor from human leukocyte

Human leukocyte cDNA library was screened to isolate cDNA clones coding for hepatocyte growth factor using cDNA from human liver as a probe. Nucleotide and deduced amino acid sequences were analyzed for two of four clones obtained. One of them contained an open reading frame coding for a polypeptide chain of 728 amino acid residues like that of cDNA clone derived from human liver. In another clone a spontaneous deletion of 15 base pairs was found within the coding sequence. When expressed transiently using COS-1 cells both clones produced protein with similar biological activity against rat hepatocyte in vitro.     

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3'- and 5'-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34,462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases was found as a highly conserved sequence.     

Isolation and characterization of mouse MUC18 cDNA gene, and correlation of MUC18 expression in mouse melanoma cell lines with metastatic ability

The cell surface adhesion molecule human MUC18 (huMUC18 or Mel-CAM) has been postulated to play a key pathogenic role in metastatic melanoma progression. To establish an immunocompetent syngeneic mouse model that would greatly facilitate our understanding of the role of MUC18 in the metastatic behavior of melanoma, we cloned and characterized the mouse MUC18 (muMUC18) cDNA gene. The gene was amplified by RT-PCR and RACE of the poly(A)+RNA isolated from the mouse melanoma cell line B16F10/Queens. The cloned muMUC18 cDNA gene contained 28 nucleotides of 5'-UTR, 908 nucleotides of 3'-UTR, and an open reading frame (ORF) of 1947 nucleotides encoding a protein of 648 amino acids, which is two amino acids longer than huMUC18. The size of the muMUC18 mRNA is about 3 kb with a shorter 3'-UTR than the huMUC18 mRNA (about 3.3 kb). Besides, the sequence in the 3' UTR of the two mRNAs is diverse with only 31% identity. The 5'-UTR and coding sequences of the muMUC18 cDNA are 72.4 and 80.6% identical to those of huMUC18, respectively. The deduced amino acid sequence of the muMUC18 cDNA is 76.2% identical to that of huMUC18. The amino acid sequences deduced from MUC18 cDNA sequences from six other mouse melanoma cell lines are identical except one to three residues, suggesting that the muMUC18 cDNA sequence determined in this report is correct. The muMUC18 protein is predicted to be slightly more acidic than the human protein. The levels of muMUC18 mRNA and protein in nine mouse melanoma cell lines were directly proportional to their ability to establish metastatic colonies in lungs of syngeneic mice. Most biological functions of the muMUC18 may be similar to the huMUC18.     

Cloning and characterization of the cDNAs for human and rabbit interleukin-1 precursor.

DNA sequence complementary to the mRNA for rabbit interleukin-1 precursor (preIL-1) has been cloned from the cDNA library constructed using partially purified poly(A)+RNA from induced rabbit alveolar macrophages by mRNA hybridization-translation assay. By using this cDNA as a probe, human IL-1 cDNA was isolated from the cDNA library prepared using poly(A)+RNA from induced HL-60 cells, a human monocyte-like cell line. The amino acid sequences of the human and rabbit preIL-1 deduced from the cDNA sequences reveal their primary structures which consists of 271 and 267 amino acid residues, respectively. The amino acid sequence is 64% conserved between human and rabbit. The difference in number of amino acid residues results from the carboxy-terminal extention of 4 amino acid residues in human preIL-1. Expression of the cloned human cDNA in E. coli yielded biologically active IL-1.     

Characterization of two mRNA species encoding human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17

Using two 33-mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of estradiol 17 beta-dehydrogenase (E2DH) and polyclonal antibodies raised against the enzyme purified from human placenta, clones were isolated from a lambda gt11 human placental cDNA library. A 327-amino acid sequence was deduced from cDNA sequencing. Two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kb while a minor one is found at approx. 2.2 kb. Primer extension and S1 nuclease analysis indicate that the major mRNA species starts 9-10 nucleotides while the minor mRNA starts 971 nucleotides upstream from the ATG initiating codon, respectively. Sequence analysis of the longest cDNA clone (2092 bp) shows that it possesses identical coding and non-coding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5' non-coding fragment hybridizes only to the 2.2 kb band, thus providing evidence for the existence of two distinct mRNA species which differ in their 5' noncoding regions. Using hp E2DH-36 cDNA as a probe for in situ hybridization of translocated chromosomes, the human E2DH gene was localized to the q11-q12 region of chromosome 17.