The complete nucleotide sequence of human liver cytochrome b5 mRNA

We have isolated and sequenced a cDNA clone corresponding to human liver cytochrome b5 mRNA. The 760 base pair (bp) sequence contains the complete coding and 3' non-translated regions plus 52 bp of 5' non-translated sequence. The derived amino acid sequence showed that the previous assignment of several amino acids was in error. In addition, the sequence of the previously unknown COOH hydrophobic region has been obtained.     

cDNA cloning of a novel cysteine protease of Plasmodium falciparum

The cDNA for a novel Plasmodium cysteine protease (falcipain-2) has been isolated from a Plasmodium falciparum cDNA library. A 602 bp fragment was amplified from P. falciparum by PCR using degenerate oligonucleotide primers. The primers were designed based upon the amino acids flanking the active site cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteases. This fragment was used to screen a P. falciparum cDNA library and isolated a 2.1 kb clone that encoded a novel cysteine protease. The sequence of the 2.1 kb clone predicted a 56 kDa protein containing a typical signal sequence, a prosequence and a 24.7 kDa mature protease with 37% identity to falcipain-1, a hemoglobin-degrading cysteine protease of P. falciparum. Northern blot analysis detected a 2.1 kb message in trophozoites. Taken together, we have isolated a novel cysteine protease of P. falciparum, which may play an important role at the late stages of the erythrocytic cycle of the parasite.     

The nucleotide sequence of a full length cDNA clone encoding rat liver urate oxidase

Recently we reported the sequence of a cDNA clone (pUOX-1), isolated from a lambda gt11 cDNA library, which encoded for rat liver urate oxidase (EC 1.7.3.3), but this clone lacked the nucleotide sequences encoding the N-terminal region for this enzyme. Using the cDNA insert from the pUOX-1 clone as a probe, we have now isolated a full length cDNA clone, pUOX-2, from a lambda gt10 library by plaque hybridization. Nucleotide sequence analysis of the pUOX-2 clone showed that it has 1379 base pairs with an open reading frame coding for 303 amino acid residues corresponding to a molecular mass of 34,931 daltons. In addition to the open reading frame the pUOX-2 contains 439 bp of 3'-untranslated and 41 bp of 5'-untranslated sequences. The consensus polyadenylation signal AATAAA precedes a stretch of poly(A)+ residues at the 3' end.     

CaMBP-10的cDNA克隆和表达及钙调素结合活性分析

采用RT PCR法 ,从中国大白菜中分离了编码CaMBP 1 0的cDNA克隆 .该cDNA全长 4 96bp ,编码 92个氨基酸 ,3′端含有 2 1 6bp的非编码区和poly A尾 .将此BP 1 0cDNA的成熟蛋白序列导入表达质粒pET1 5b并转化至大肠杆菌E .coliBL2 1 (DE3)condonplus RIL进行表达 .以免疫印迹和钙调素结合分析法对重组BP 1 0进行鉴定 ,证明其保持了与天然BP 1 0相同的钙调素结合活性 .氨基酸和核苷酸序列分析结果显示 ,它与植物转脂蛋白高度同源 ,特别是含有 8个保守半胱氨酸 .BP 1 0与转脂蛋白之间具极为相似的理化性质如分子量、等电点、热稳定性等 .据此认为 ,CaMBP 1 0是转脂蛋白家族的新成员 ,Ca2 + CaM信号系统可能参与植物转脂蛋白功能的调节     

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.     

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.     

Isolation and sequence determination of cDNA encoding P2 protein of human peripheral myelin.

A full length cDNA of P2 protein of peripheral myelin has been isolated from a cDNA library of human fetus spinal cord. The clone is 2150 base pairs (bp) in length and contains a 393 bp open reading frame encoding a polypeptide of 131 residues. The deduced amino acid sequence is highly homologous to P2 protein from other species.     

Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex

We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.     

Soluble and membrane-bound forms of cytochrome b5 are the products of a single gene in chicken

In order to determine the relationship of the soluble cytochrome b5 found in erythrocytes to the membrane-bound form found in other tissues, a cDNA clone encoding cytochrome b5 in chicken erythrocytes was isolated by using mixed oligonucleotides based on a partial amino acid sequence of the protein. Complete nucleotide sequence identity between the erythrocyte cDNA and the sequence of a cDNA clone of the liver protein suggests that they are transcribed from the same gene. The isolation and structural analysis of genomic clones was also consistent with the presence of only one cytochrome b5 gene in chicken. These results suggest that the formation of soluble erythrocyte cytochrome b5 occurs by proteolytic processing of the membrane-bound form. Thus, previous reports indicating that the carboxyl terminal amino acid residue of the erythrocyte form differs from the corresponding residue of the membrane-bound form may suggest the existence of a novel post-translational modification.     

Sexually dimorphic and ontogenetic expression of dmrt1, cyp19a1a and cyp19a1b in Gobiocypris rarus

Fish have diverse sex determination and differentiation. DMRT1 and aromatase are conserved in the phyla and play pivotal roles in sex development. Gobiocypris rarus is a small fish used as a model in aquatic toxicology in China and has been used to study the effects of environmental endocrine disruptors on gene expression, but its sexual development remains elusive. Here, we report the full-length cDNA of G. rarus dmrt1 and its expression along with the expression of cyp19a1a and cyp19a1b, two genes encoding gonad and brain type aromatases, in adults and during ontogenesis. Both cyp19a1a and dmrt1 are expressed in the ovary and testis but show sexual dimorphism. Expression of cyp19a1a in the ovary is higher than in testes and dmrt1 follows the opposite pattern. Juvenile gonad histology changes at 15 days after hatching. The dimorphic expression of dmrt1 and cyp19a1a appears from 5 days after hatching, which is earlier than histological change. cyp19a1b is expressed coordinately with cyp19a1a until 15 days after hatching. These results show that dmrt1 and cyp19a1a play important roles in sex determination and sex differentiation in G. rarus.     

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.     

Molecular characterization and sex-specific tissue expression of estrogen receptor alpha (esr1), estrogen receptor betaa (esr2a) and ovarian aromatase (cyp19a1a) in yellow perch (Perca flavescens)

Yellow perch (Perca flavescens) exhibits an estrogen-stimulated sexual size dimorphism (SSD) wherein females grow faster and larger than males. To aid in the examination of this phenomenon, the cDNA sequences encoding estrogen receptor-alpha (esr1), estrogen receptor-betaa (esr2a) and ovarian aromatase (cyp19a1a) for the teleost yellow perch were obtained. Several tissues were analyzed from both male and female adult yellow perch for sex-specific tissue expression. The full length cDNAs of yellow perch esr1, esr2a and cyp19a1a consist of 3052 bp, 2462 bp and 1859 bp with open reading frames encoding putative proteins of 576 amino acids, 555 amino acids and 518 amino acids, respectively. Esr1 and esr2a expression was highest in female ovary and liver tissues with low to moderate expression in other tissues. Esr2a showed a more global tissue expression pattern than esr1, particularly in males but also in females. Cyp19a1a expression was highest in both male and female spleen tissue and oocytes with moderate expression in male pituitary and gill tissue. Cyp19a1a expression was moderately high in female liver tissue with undetectable expression in male liver tissue, suggesting its involvement in sexually dimorphic growth. These sequences are valuable molecular tools that can be used in future studies investigating estrogen mechanisms and actions, such as SSD, in yellow perch.     

Structural analysis of human complement protein H: homology with C4b binding protein, beta 2-glycoprotein I, and the Ba fragment of B2

We report here a partial primary structure for human complement protein H. Tryptic peptides comprising 27% of the H molecule were isolated by conventional techniques and were sequenced (333 amino acid residues). Several mixed-sequence oligonucleotide probes were constructed, based on the peptide sequence data, and were used to screen a human liver cDNA library. The largest recombinant plasmid (pH1050), which hybridized with two probes, was further characterized. The cDNA insert of this plasmid contained coding sequence (672 bp) for 224 amino acids of H. The 3' end of this clone had a polyadenylated tail preceded by a polyadenylation recognition site (ATTAAA) and a 3'-untranslated region (229 bp). Four regions of internal homology, each about 60 amino acids in length, were observed in the derived protein sequence from this cDNA clone, and a further seven from the tryptic peptide sequences. The consensus sequence for each of the repetitive units of H was four cysteines, two prolines, three glycines, one tryptophan, and two tyrosines/phenylalanines. Based on the mole percent values for each of these amino acids, it is likely that H is composed of about 20 repetitive units of this nature. Furthermore, the repetitive unit of H shows pronounced homology with the Ba fragment of B, the C4b binding protein, and beta 2-glycoprotein I. Therefore, it seems that at least portions of these proteins have evolved from a common ancestral DNA element.