Complete nucleotide sequence of the high molecular weight human IGF-I mRNA

IGF-I gene expression in mammals typically results in multiple mRNA species ranging in size between 0.7 and 7.6 kb. The smaller mRNA species have largely been characterized by the analysis of nearly full-length cDNAs. This report describes the first complete sequence of the prominent high molecular weight (7.6 kb) IGF-I mRNA species. Isolation and nucleotide sequence analysis of cDNA clones from human adult liver and uterus leiomyoma cDNA libraries resulted in a 7236 bp long sequence followed by a poly(A) tail. The sequence data, in combination with structural analysis of the human IGF-I gene, show that the 7.6 kb human IGF-I mRNA contains 6611 bp of untranslated 3' terminal sequence derived from a single exon. Alternate employment of two polyadenylation signals within the sequence transcribed from this exon generates two mRNAs of 1.1 and 7.6 kb.     

Molecular cloning of the alcohol/hydroxysteroid form (hSTa) of sulfotransferase from human liver.

A cDNA encoding the human alcohol/hydroxysteroid sulfotransferase (h-ST-a), which catalyzes the sulfo-conjugation of many drugs and hormones, was isolated from a human liver cDNA library using a rat STa (rSTa) cDNA probe. The cDNA, designated as hSTa, consists of 1069 base pairs (bp) and contains an 855-nucleotide open reading frame beginning at nucleotide 65, which encodes a 285 amino acid polypeptide of 33.76 kDa. A second cDNA clone (1563 bp) was truncated 5' at nucleotide 231 (lacking the first 15 amino acids) with identical coding region, however, it had a much longer 3' untranslated region (UTR). Both clones contained a short segment of poly(A)+ tail. Northern blot analysis of an adult human liver showed that there are at least 2 mature mRNA with sizes ranging from approximately 1.1 kb to 1.7 kb, verifying the authenticity of the obtained cDNA clones. From the sequence alignment, the hSTa shares 62%/74%, 39%/59%, 35%/48%, 36%/54% identity with rSTa, rSTp (phenol), rSTe (estrogen), and bovine STe (bSTe) at the deduced amino acid and DNA levels, respectively, indicating that there are at least three subfamilies (alcohol, phenol and estrogen) of genes that encode for sulfotransferases in mammals.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

Molecular cloning and nucleotide sequence of the cDNA for human liver glutamate dehydrogenase precursor

Two cDNA clones (lambda GDHh1 and lambda GDHn61) for glutamate dehydrogenase (GDH) were isolated from a human liver cDNA library in lambda gt11. The clone, lambda GDHh1, was isolated from the library using a synthetic 45mer oligodeoxy-ribonucleotide, the sequence of which was derived from the known amino acid sequence near the NH2-terminus of human liver GDH. Subsequently, lambda GDHn61 was isolated from the same library using lambda GDHh1 as a probe. The inserts of both clones contained an overlapping cDNA sequence for human liver GDH, consisting of a 5'-untranslated region of 70 bp, an open reading frame of 1677 bp, a 3'-untranslated region of 1262 bp and a 15 base poly(A) tract. The predicted amino acid sequence revealed that the human liver GDH precursor consisted of a total of 558 amino acid residues including the NH2-terminal presequence of 53 amino acids. The sequence deduced for the mature enzyme showed 94% homology to the previously reported amino acid sequence of human liver GDH.     

The isolation and characterisation of cDNA and genomic clones for human lecithin: cholesterol acyltransferase

The protein sequencing of tryptic peptides from purified human lecithin: cholesterol acyltransferase (LCAT) identified sufficient amino-acid sequence to construct a corresponding mixed oligonucleotide probe. This was used to screen an adult human cDNA liver library, from which incomplete cDNA clones were isolated. The DNA sequence of these clones allows the prediction of the entire amino-acid sequence of the mature LCAT enzyme. The mature protein consists of 416 amino acids and contains several marked stretches of hydrophobic residues and four potential glycosylation sites. The cDNA probe detects LCAT mRNA sequences approx. 1500 bases long in human liver, but not intestine, RNA. The cDNA probe was used to isolate LCAT genomic recombinants from a human genomic library. Southern blotting data, and restriction site mapping, suggest that there is a single human LCAT structural gene between 4.3 and 5.5 kb in size.     

Cloning and characterization of a cDNA that encodes a 70-kDa novel human thyroid autoantigen

cDNA clones were isolated by screening a human thyroid carcinoma lambda gt11 library with immunoglobulins purified from serum of a patient with autoimmune Graves' disease. One clone (ML8) containing a 1.25-kilobase (kb) insert hybridized with a single 2.0-kb poly(A+) mRNA in human thyroid and lymphocytes but not in human brain, liver, kidney, or muscle. In addition, this probe also hybridized with a single 2.0-kb poly(A+) mRNA from a rat thyroid cell line (FRTL-5). An apparently full length 2,074-base pair (bp) human cDNA was obtained and sequenced. The nucleotide sequence of the 2,074-bp cDNA includes a 5'-noncoding sequence of 17 bp, a 1827-bp open reading frame, and a 222-bp 3'-noncoding sequence. The canonical polyadenylation signal AATAAA is present 18 bp upstream of the poly(A) tail. This cDNA encodes a 69,812-dalton protein with two potential N-linked glycosylation sites and at least one potential membrane spanning domain. Immunoprecipitation of the in vitro translated protein by sera from several patients with Graves' disease argues that the 69,812-dalton protein is an autoantigen.     

The isolation of cDNA clones for human apolipoprotein E and the detection of apoE RNA in hepatic and extra-hepatic tissues.

We have isolated cDNA clones coding for apolipoprotein E (apoE) from a cDNA library prepared from adult human liver mRNA. Mixtures of 128 different oligonucleotides, 17 residues long were synthesised to be complementary to regions of the mRNA corresponding to amino acids 1-6 and 151-156. Five independent apoE clones were selected by direct screening of 5000 recombinants with the two oligonucleotide mixtures. Two overlapping clones contain the 3'-untranslated sequence, the entire coding sequence and an additional 30 bases 5' to the amino terminus of the mature protein. The DNA sequence has been determined spanning the known sites of amino acid substitutions which account for the observed protein polymorphism of apoE. Using the clones as probes in Northern blot analysis of total human liver and kidney RNAs and leucocyte poly(A)+ RNA we have detected a single species of mRNA in liver and kidney of 1.2 kb and two larger species in leucocyte RNA. The level of expression of the mRNA in kidney is approximately 10% of that in liver while the level of apoE RNA sequences in the leucocytes is less than 1% of that in the liver.     

Gene structure of human and mouse methylenetetrahydrofolate reductase (MTHFR)

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine. A human cDNA for MTHFR, 2.2 kb in length, has been expressed and shown to result in a catalytically active enzyme of approximately 70 kDa. Fifteen mutations have been identified in the MTHFR gene: 14 rare mutations associated with severe enzymatic deficiency and 1 common variant associated with a milder deficiency. The common polymorphism has been implicated in three multifactorial diseases: occlusive vascular disease, neural tube defects, and colon cancer. The human gene has been mapped to chromosomal region 1p36.3 while the mouse gene has been localized to distal Chromosome (Chr) 4. Here we report the isolation and characterization of the human and mouse genes for MTHFR. A human genomic clone (17 kb) was found to contain the entire cDNA sequence of 2.2 kb; there were 11 exons ranging in size from 102 bp to 432 bp. Intron sizes ranged from 250 bp to 1.5 kb with one exception of 4.2 kb. The mouse genomic clones (19 kb) start 7 kb 5′ exon 1 and extend to the end of the coding sequence. The mouse amino acid sequence is approximately 90% identical to the corresponding human sequence. The exon sizes, locations of intronic boundaries, and intron sizes are also quite similar between the two species. The availability of human genomic clones has been useful in designing primers for exon amplification and mutation detection. The mouse genomic clones will be helpful in designing constructs for gene targeting and generation of mouse models for MTHFR deficiency. Received: 28 January 1998 / Accepted: 9 April 1998     

The sedlin gene for spondyloepiphyseal dysplasia tarda escapes X-inactivation and contains a non-canonical splice site

Mutations in the sedlin gene cause spondyloepiphyseal dysplasia tarda (SEDT), a rare X-linked chondrodysplasia. Affected males suffer short stature, deformation of the spine and hips, and deterioration of intervertebral discs with characteristic radiographic changes in the vertebrae. We have sequenced two full-length cDNA clones corresponding to the human sedlin gene. The longest cDNA is 2836 bp, containing a 218 bp 5' untranslated region, a 423 bp coding region, and a 2195 bp 3' untranslated region. The second cDNA does not contain exon 2, suggesting alternative splicing. Sedlin was finely mapped in Xp22.2 by Southern blot analysis on a yeast artificial chromosome/bacterial artificial chromosome map. Comparison of the cDNA sequence and genomic sequence identified six sedlin exons of 67, 142, 112, 147, 84, and 2259 bp. The corresponding introns vary in size from 339 to 14,061 bp. Splice site sequences for four of the five introns conform to the GT/AG consensus sequences, however, the splice site between exons 4 and 5 displays a rare non-canonical splice site sequence, AT/AC. Northern blot analysis showed expression of the sedlin gene in all human adult and fetal tissues tested, with the highest levels in kidney, heart, skeletal muscle, liver, and placenta. Four mRNA sizes were detected with the major band being 3 kb and minor bands of 5, 1.6, and 0.9 kb (the smallest product may reflect a sedlin pseudogene). Sedlin is expressed from both the active and the inactive human X chromosomes helping to explain the recessive nature and consistent presentation of the disease. Human sedlin shows homology to a yeast gene, which conditions endoplasmic reticulum/golgi transport. Characterization of the human sedlin cDNA and determination of the sedlin gene structure enable functional studies of sedlin and elucidation of the pathogenesis of SEDT.     

Isolation, expression and characterization of a human apolipoprotein B 100-specific cDNA clone

The isolation and characterization of a human apolipoprotein B 100-specific cDNA clone (lambda gt-B1) containing a 1321 base pairs (bp) spanning insert is described. It encodes the 3'-nontranslated 281 bp long region up to the polyadenylation site and 1040 bp of the C-terminal coding region of 345 amino-acid residues of human apo B 100 and the stop codon. The lambda gt-B1 cDNA clone has been isolated from a human hepatoma cDNA expression library by immunoscreening using affinity-purified polyclonal anti apo B 100 antibodies. The nucleotide sequence of the apo B 100 insert has been determined. A part of the polypeptide sequence derived from this nucleotide sequence was identical with the amino-acid sequence obtained by protein sequencing of a purified cyanogen bromide fragment of apo B 100. The fusion protein consisting of beta-galactosidase and the 345 amino-acid residue long C-terminus of apo B 100 had an apparent molecular mass of 148 kDa in NaDodSO4 polyacrylamide gel electrophoresis. In Northern blot hybridization analysis the insert of the apo B 100-cDNA clone hybridized to a 20 to 22 kb mRNA from adult human liver.     

Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.     

Complementary DNA derived structure of the amino-terminal domain of human apolipoprotein B and size of its messenger RNA transcript

In this paper the sequence of a 5.2-kilobase (kb) cDNA covering the amino-terminal domain of human apolipoprotein B-100 (apoB-100) is reported. The cDNA-derived protein sequence provides the primary structure of 1748 amino acids. This segment of apoB-100 is more hydrophilic than hydrophobic and contains short stretches of predicted helical and beta structures that are interrupted by beta turns. Blotting analysis of RNA isolated from fetal human and adult monkey tissues and various human cell lines showed synthesis of apoB mRNA by liver and intestine and by cells of hepatic (HepG2) and intestinal (Caco-2) origin. The isolation and characterization of overlapping cDNA clones, which provide a nearly full-length copy of human apoB-100, are also reported. From the length of these clones the size of the cytoplasmic apoB mRNA is estimated to be 14.0 kb and codes for a protein of approximately 512,000 daltons.     

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

Molecular cloning of the acid-labile subunit of the rat insulin-like growth factor binding protein complex.

The insulin-like growth factors, IGF-I and IGF-II, circulate in both humans and rats as part of a 125-150 kDa complex comprising IGFs, the IGF binding protein IGFBP-3, and an acid-labile subunit. Clones encoding rat acid-labile subunit have been isolated from a rat liver cDNA library probed with a human acid-labile subunit cDNA. Two overlapping clones encode a leucine-rich protein of 576 amino acids preceded by a 27-residue signal sequence, with 78% homology to the human acid-labile subunit. Northern analysis of mRNA from adult rat brain, kidney, heart, lung, spleen, muscle and liver shows a major species of about 4.4 kb and minor bands of about 2 kb, 1.4 kb and 1 kb. The tissue distribution of this protein may therefore be wider than previously recognized.     

Isolation and characterization of human cDNA clones encoding the alpha and the alpha' subunits of casein kinase II

Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two alpha or alpha' subunits (or one of each) and two beta subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell lambda gt10 library using cDNA clones isolated from Drosophila melanogaster [Saxena et al. (1987) Mol. Cell. Biol. 7, 3409-3417]. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 clone was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells [Meisner et al. (1989) Biochemistry 28, 4072-4076]. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 bp (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of alpha and alpha' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the alpha and alpha' subunits of casein kinase II. Microsequence data determined from separated preparations of bovine casein kinase II alpha subunit and alpha' subunit [Litchfield et al. (1990) J. Biol. Chem. 265, 7638-7644] confirmed that hT4.1 encoded the alpha subunit and hT9.1 encoded the alpha' subunit. These studies show that there are two distinct catalytic subunits for casein kinase II (alpha and alpha') and that the sequence of these subunits is largely conserved between the bovine and the human.     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。     

Isolation and characterization of a cDNA for rat liver cysteine dioxygenase

Cysteine dioxygenase is a key enzyme of cysteine metabolism in mammals. The cDNA clones for rat liver cysteine dioxygenase were isolated by immunological screening and plaque hybridization from a rat liver cDNA library. The longest clone contained an insert of 1458 bp and encoded a polypeptide of 200 amino acids. The clone included the corresponding nucleotide sequence to amino acid sequences obtained from four lysyl endopeptidase-digested fragments of purified rat liver cysteine dioxygenase. The calculated molecular weight of rat liver cysteine dioxygenase was 23,025. Northern blot analysis revealed a single cysteine dioxygenase mRNA species of about 1.7 kb. A computer homology search indicated that this protein showed no homology with any known protein.