Bovine lactoferrin mRNA: sequence, analysis, and expression in the mammary gland.

The mRNA sequence for bovine lactoferrin expressed in the mammary gland was determined by sequencing three over lapping cDNA clones and by direct sequencing of the mRNA. The mRNA (2351 bases) codes for a 708 amino acid protein with a 19 amino acid signal peptide immediately preceding a sequence identical to the N-terminal 40 amino acids reported for bovine lactoferrin. A putative destabilizing sequence (AUUUA) was identified in the 3'-untranslated region. The nucleic acid sequence and deduced amino acid sequence are highly homologous with other transferrin family members. Lactoferrin mRNA concentrations in bovine mammary tissue were quite low two days before parturition and during lactation but were high three days after the cessation of milking, a sharp contrast from the pattern of regulation of the other milk proteins.     

Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.

Triacylglycerol hydrolase mobilizes stored triacylglycerol some of which is used for very-low-density lipoprotein assembly in the liver. A full-length cDNA coding for a human triacylglycerol hydrolase (hTGH) was isolated from a human liver cDNA library. The cDNA has an open reading frame of 576 amino acids with a cleavable 18-amino-acid signal sequence. The deduced amino acid sequence shows that the protein belongs to the carboxylesterase family. The hTGH was highly expressed in Escherichia coli as a 6xHis-tagged fusion protein, with the tag at the N-terminus in place of the signal peptide. However, the expressed protein was insoluble and inactive. Expression was confirmed by immunoblotting and N-terminal amino acid sequencing of the purified protein. Expression of hTGH with its native signal sequence and a C-terminal 6xHis-tag in Sf9 cells using the baculovirus expression system yielded active enzyme. N-terminal amino acid sequencing of the purified expressed protein showed correct processing of the signal peptide. The enzyme also undergoes glycosylation within the endoplasmic reticulum lumen. The results suggest that hTGH expressed in insect cells is properly folded. Therefore, baculovirus expression of hTGH and facile purification of the His-tagged enzyme will allow detailed characterization of the structure/activity relationship.     

Cloning and expression of the growth hormone-dependent insulin-like growth factor-binding protein

N-terminal as well as internal amino acid sequence data were obtained from the GH dependent, insulin-like growth factor (IGF) binding protein, BP-53, purified from human plasma. Based on these sequence data, full-length cDNA clones of BP-53 have been isolated, and the complete deduced sequence of BP-53 determined. This sequence contains a 27 amino acid putative signal sequence followed by a mature protein of 264 amino acids containing 18 cysteine residues clustered near the N- and C-terminus. The deduced protein sequence of BP-53 has 33% amino acid identity including conservation of all 18 cysteine residues with the recently cloned BP-28, a smaller human IGF-binding protein identified in amniotic fluid and also secreted by the cell line HEP G2. Expression of the cloned BP-53 cDNA in mammalian tissue culture cells results in secretion of the protein into the culture medium. This expressed protein is identical to plasma-derived BP-53 in its immunoreactivity, high affinity binding of IGF-I and IGF-II, and mobility on sodium dodecyl sulfate gel electrophoresis.     

Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life.

The 11-4 p53 cDNA clone failed to transform primary rat fibroblasts when cotransfected with the ras oncogene. Two linker insertion mutations at amino acid 158 or 215 (of 390 amino acids) activated this p53 cDNA for transformation with ras. These mutant cDNAs produced a p53 protein that lacked an epitope, recognized by monoclonal antibody PAb246 (localized at amino acids 88 to 110 in the protein) and preferentially bound to a heat shock protein, hsc70. In rat cells transformed by a genomic p53 clone plus ras, two populations of p53 proteins were detected, PAb246+ and PAb246-, which did or did not bind to this monoclonal antibody, respectively. The PAb246- p53 preferentially associated with hsc70, and this protein had a half-life 4- to 20-fold longer than free p53 (PAb246+). These data suggest a possible functional role for hsc70 in the transformation process. cDNAs for p53 derived from methylcholanthrene-transformed cells transform rat cells in cooperation with the ras oncogene and produce a protein that bound with the heat shock proteins. Recombinant clones produced between a Meth A cDNA and 11-4 were tested for the ability to transform rat cells. A single amino acid substitution at residue 132 was sufficient to activate the 11-4 p53 cDNA for transformation. These studies have identified a region between amino acids 132 and 215 in the p53 protein which, when mutated, can activate the p53 cDNA. These results also call into question what the correct p53 wild-type sequence is and whether a wild-type p53 gene can transform cells in culture.     

Nucleotide sequence of the p53 cDNA of beluga whale (Delphinapterus leucas)

The cDNA (DNA complementary to RNA) of the p53 gene of the beluga whale (Delphinapterus leucas) was sequenced by the method of 5'- and 3'-rapid amplification of cDNA ends (RACE) with the cDNA made for the RNA obtained from fresh peripheral blood leukocytes isolated from two animals. Primers for the RACE method were synthesized based on the sequence of the DNA of beluga whale corresponding to exon 5 of the human p53 gene, which was determined after amplification of the DNA isolated from the liver from a beluga whale by using a pair of primers for the human sequence. The sequenced cDNA had a 2150-nucleotide length and contained the whole region corresponding to human exons 1 through 11. The reading frame was 1164 bp (base pair) long and began in exon 2 and ended in exon 11, coding for a 387-amino acid protein. The nucleotide sequence of the reading frame showed high similarity over 85% with pig, sheep, cow, and human genes. The similarities with the former two animals at the amino acid level were also more than 85%. Lower similarity of the beluga whale p53 gene was also found with those of lower tetrapods, fish and invertebrates.     

Molecular cloning of a cDNA and assignment of the C-terminal of sarcotoxin IA, a potent antibacterial protein of Sarcophaga peregrina.

A previous paper described the complete amino acid sequences of sarcotoxins IA, IB and IC, which are a group of potent antibacterial proteins with almost identical primary structures produced by Sarcophaga peregrina (fleshfly) larvae [Okada & Natori (1985) J. Biol. Chem. 260, 7174-7177]. The present paper describes the cDNA cloning and complete nucleotide sequencing of a cDNA clone for sarcotoxin IA. The C-terminal amino acid residue of sarcotoxin IA deduced from the nucleotide sequence was glycine, whereas it was found to be arginine by amino acid sequencing of purified sarcotoxin IA. Analysis of the elution profiles on h.p.l.c. of the synthetic derivatives of sarcotoxin IA showed that the C-terminal amino acid residue of authentic sarcotoxin IA is amidated arginine, which is probably produced by enzymic cleavage of terminal glycine.     

Molecular cloning, sequencing and expression in Escherichia coli of the 25-kDa growth-related protein of Ehrlich ascites tumor and its homology to mammalian stress proteins

The growth-related 25-kDa protein (p25) of Ehrlich ascites tumor (EAT) has been characterized by molecular cloning and sequencing of cDNA clones detected by hybridization with oligonucleotide probes synthesized according to the amino acid sequence of a tryptic peptide of p25. Detection of p25 mRNA in EAT of the exponential growth phase and of the stationary phase using cDNA-derived RNA probes demonstrated that the abundance of p25 mRNA is also growth-related. High-level expression of p25 in Escherichia coli has been established by oligonucleotide-directed mutagenesis of cDNA and insertion of the mutated cDNA into a T7-promoter expression vector. Recombinant p25 from the expressed cDNA sequence has been shown to comigrate with EAT p25 in electrophoresis and to react with antibodies against the EAT p25. On the amino acid level, p25 shows about 80% sequence homology to the human stress protein hsp27. Furthermore, p25 has similar isoforms of phosphorylation as demonstrated for small mammalian stress proteins from rat and human. From the results obtained, it is concluded that p25 is a mammalian stress protein, the abundance of which is related to growth characteristics of the Ehrlich ascites tumor.     

VCP, the mammalian homolog of cdc48, is tyrosine phosphorylated in response to T cell antigen receptor activation.

Activation of T cells through the T cell antigen receptor (TCR) results in the rapid tyrosine phosphorylation of a number of cellular proteins, one of the earliest being a 100 kDa protein. We have sought to identify this 100 kDa substrate by partially purifying the protein by antiphosphotyrosine (APT) affinity purification, in order to obtain amino acid sequence data and, using this information, to isolate the cDNA clone encoding the molecule. We report here that the amino acid sequence data showed pp100 to be the murine equivalent of porcine valosin containing protein (VCP), a finding confirmed from the cloning and sequencing of the murine pp100 cDNA. Sequence analysis has shown VCP to be a member of a family of ATP binding, homo-oligomeric proteins, and the mammalian homolog of Saccharomyces cerevisiae cdc48p, a protein essential to the completion of mitosis in yeast. We also provide proof that both endogenous and expressed murine VCP are tyrosine phosphorylated in response to T cell activation. Thus we have identified a novel component of the TCR mediated tyrosine kinase activation pathway that may provide a link between TCR ligation and cell cycle control.     

一个鼻咽癌相关EST的鉴定及其全长cDNA序列分析

鼻咽癌是我国南方及东南亚地区常见的恶性肿瘤之一.通过对鼻咽癌染色体高频率杂合性丢失区域3p21的表达序列标签(expressedsequencetag,EST)进行同源性比较分析,运用逆转录聚合酶链式反应的方法,筛选到一个在41.18%(14/34)的鼻咽癌活检组织及20.0%(1/5)的鼻咽癌细胞系中表达下调的ESTBG772301;并用Northern杂交方法,检测了该EST在多种正常成人组织中的表达状况及其所代表基因的转录本大小.在此基础上,对该EST来源的cDNA克隆(IMAGE:4839190)进行直接测序,获得了一个全长为2377bp的新cDNA序列;经生物信息学分析,发现它与已知基因序列无明显同源性,属于一个新基因,定位于染色体3p21.3,被命名为鼻咽癌表达下调基因(NPCEDRG,GenBank登录号:AF538150).其编码的蛋白质含169个氨基酸,与一个已报道的在进化上相对保守、功能未知的人类蛋白Nicolin1(简称NICN1)N端170个氨基酸残基的序列同源性为97%,但缺少NICN1蛋白C端43个氨基酸残基,可能是nicolin1基因不同剪接本的编码产物.     

Cloning, mapping and association study with carcass traits of the porcine SDHD gene

A 1320-bp cDNA containing the full coding region of the porcine succinate dehydrogenase complex, subunit D (SDHD) gene was obtained by random sequencing of clones from a Chinese Tongcheng pig 55-day fetal longissimus dorsi muscle cDNA library. Analysis of the SDHD gene across the INRA-University of Minnesota porcine radiation hybrid panel indicated close linkage with microsatellite marker SW2401, located on SSC9p21. The open reading frame of this cDNA covers 480 bp and encodes 159 amino acids. The deduced porcine amino acid sequence showed greater similarity with human and bovine protein sequences than with those from mouse and rat. The BLAST analysis of the porcine SDHD to NCBI identified Unigene Cluster Ssc.2586. Possible single nucleotide polymorphisms (SNP) were identified by alignment of expressed sequence tags in the cluster. The polymerase chain reaction (PCR) single strand conformation polymorphism, sequencing, and PCR restriction fragment length polymorphism were used to confirm and detect a synonymous polymorphic MboI site within the open-reading frame. Allele frequencies of this SNP were investigated in two commercial and five Chinese local pig breeds. These five Chinese breeds had very high frequencies for one allele, whereas frequencies of both alleles were intermediate in Large White and Duroc. An association analysis suggested that different SDHD genotypes have significant differences in loin-muscle area (P < 0.01).     

p53 gene expression is modulated by endocrine disrupting chemicals in the hermaphroditic fish, Kryptolebias marmoratus

The full-length of cDNA of tumour suppressor gene p53 from the self-fertilizing fish Kryptolebias marmoratus (Km-p53) was determined using molecular cloning and rapid amplification of cDNA ends (RACE). The Complete cDNA sequences of K. marmoratus (Km-p53) gene was 1.8 kb in length. K. marmoratus p53 amino acid sequence showed a high degree of homology with the sequences from fishes, amphibians, and mammals. Although basal level of expression of Km-p53 mRNA was low, all the studied tissues showed some level of expression. After exposure of K. marmoratus to endocrine disrupting chemicals (EDCs) such as bisphenol A, 4-nonylphenol, and 4-tert-octylphenol, Km-p53 expression was significantly increased within 3 h of exposure in juveniles. However, expression was down-regulated by exposure to most of the EDCs when measured at 96 h in adult fish. In adult fish, suppressive effect of EDCs was more pronounced in liver as compared to other tissues. These findings suggest that Km-p53 gene would be involved in cellular defense mechanism in early stage of exposure to EDCs and long-term exposure may suppress its expression. It may be possible that the suppression of p53 by EDCs may predispose the host to environmental chemical carcinogenesis.     

Sequence and genomic structure of the human adult skeletal muscle sodium channel alpha subunit gene on 17q.

The amino acid sequence of the sodium channel alpha subunit from adult human skeletal muscle has been deduced by cross-species PCR-mediated cloning and sequencing of the cDNA. The protein consists of 1836 amino acid residues. The amino acid sequence shows 93% identity to the alpha subunit from rat adult skeletal muscle and 70% identity to the alpha subunit from other mammalian tissues. A 500 kb YAC clone containing the complete coding sequence and two overlapping lambda clones covering 68% of the cDNA were used to estimate the gene size at 35 kb. The YAC clone proved crucial for gene structure studies as the high conservation between ion channel genes made hybridization studies with total genomic DNA difficult. Our results provide valuable information for the study of periodic paralysis and paramyotonia congenita, two inherited neurological disorders which are caused by point mutations within this gene.     

Amino acid sequence of human histidine-rich glycoprotein derived from the nucleotide sequence of its cDNA

A lambda gt 11 library containing cDNA inserts prepared from human liver mRNA has been screened with an affinity-purified antibody to human histidine-rich glycoprotein (HRG) and then with a restriction fragment isolated from the 5' end of the largest cDNA insert obtained by antibody screening. A number of positive clones were identified and shown to code for HRG by DNA sequence analysis. A total of 2067 nucleotides were determined by sequencing 3 overlapping cDNA clones, which included 121 nucleotides of 5'-noncoding sequence, 54 nucleotides coding for a leader sequence of 18 amino acids, 1521 nucleotides coding for the mature protein of 507 amino acids, a stop codon of TAA, and 352 nucleotides of 3'-noncoding sequence followed by a poly(A) tail of 16 nucleotides. The length of the noncoding sequence of the 3' end differed in several clones, but each contained a polyadenylylation or processing sequence of AATAAA followed by a poly(A) tail. More than half of the amino acid sequence of HRG consisted of five different types of internal repeats. Within the last 3 internal repeats (type V), there were 12 tandem repetitions of a 5 amino acid segment with a consensus sequence of Gly-His-His-Pro-His. This repeated portion, referred to as a "histidine-rich region", contained 53% histidine and showed a high degree of similarity to a histidine-rich region of high molecular weight kininogen.     

Differential responses of p56lyn and p53lyn, products of alternatively spliced lyn mRNA, on stimulation of B-cell antigen receptor.

We previously cloned a lyn cDNA-encoding 56-kd Src-like protein-tyrosine kinase, p56lyn. Anti-Lyn antibodies raised against a sequence of 95 amino acids (Arg-25 to Ala-119 of p56lyn) recognized two species of the protein, p56lyn and p53lyn. V8 proteinase analysis showed that p53lyn differs only slightly from p56lyn. Analysis of mRNA from B lymphocytes by the polymerase chain reaction indicated the presence of two forms of alternatively spliced lyn mRNA. Nucleotide sequencing of the corresponding cDNAs revealed that these two forms of lyn mRNA differ in the presence and absence of a 63 nucleotides sequence near the 5'-terminus of the coding region; 21 amino acid residues (Pro-23 to Arg-43 or Val-24 to Pro-44) of p56lyn were tentatively concluded to be missing in p53lyn. On cross-linking of the membrane-bound IgM (mIgM) on the surface of B lymphocytes, the kinetics of down-regulations of the two Lyn proteins demonstrated to be associated with the mIgM antigen receptor were found to be different. This observation suggests that the amino terminal proximal sequence of the Lyn protein is important for determining its mode of interaction with mIgM.     

Correction of the cDNA-derived protein sequence of prostatic spermine binding protein: pivotal role of tandem mass spectrometry in sequence analysis

Spermine binding protein (SBP) is a rat ventral prostate protein that binds various polyamines, and the level of this protein and its mRNA is regulated by androgens. Previously, the cDNA for SBP was cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from partial amino acid sequencing of the purified protein were consistent with the amino acid sequence deduced from the cDNA. However, the amino terminus of the protein was blocked, and therefore, direct protein sequence information confirming the cDNA reading frame of this region could not be obtained by Edman degradation. We have now employed an integrated approach using fast atom bombardment mass spectrometry, tandem mass spectrometry, and conventional sequencing methodologies to establish the amino-terminal sequence of the protein and to identify an amino acid sequence (35 residues) present in the purified protein but missing from the amino acid sequence deduced from cDNA clones for this protein. The missing piece of cDNA corresponds to an exon found in mouse genomic clones for a protein similar to rat SBP. Therefore, the cDNA clones for rat SBP may represent splicing variants that lack the sequence information of one exon. The blocked amino terminus of the protein was identified as 5-oxopyrrolidine-2-carboxylic acid. Mass spectrometry also provided evidence regarding glycosylation of the protein. The first of two potential glycosylation sites clearly carries carbohydrate; the second site is, at most, only partially glycosylated.     

Polycalin (chlorophyllid A binding protein): a novel, very large fluorescent lipocalin from the midgut of the domestic silkworm Bombyx mori L

We studied a protein from the midgut of the silkworm Bombyx mori characterized by its ability to bind the prosthetic group of chlorophyll, that confers fluorescent properties to this protein. Several techniques, 2D electrophoresis purification, MS-MS and Maldi-TOF peptide sequencing, RT-PCR and nucleotide sequencing were used to obtain the nucleotide sequence and the deduced amino acid sequence. The coding sequence was compared to the gene sequence to define the number and size of introns and exons.The gene spanned 45.5 kb of DNA and consisted of 46 exons. The cDNA encoded a protein of 2721 amino acids.The protein was identified as a lipocalin with novel features. Most lipocalins are proteins with high affinity to small lipophilic molecules, with a molecular size in the 25 kDa range and a well conserved tertiary structure. The apoprotein described here revealed 15 lipocalin like structures, in line. We called this protein a polycalin (pentadecacalin).