Molecular cloning of the complementary DNA for human tumor necrosis factor

Tumor necrosis factor (TNF) is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF was purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and showed cytotoxic and cytostatic activities against various human tumor cell lines. The amino acid sequence was determined for the amino terminal end of the purified protein, and oligodeoxyribonucleotide probes were synthesized on the basis of this sequence. Complementary DNA (cDNA) encoding human TNF was cloned from induced HL-60 messenger RNA and was confirmed by hybrid-selection assay, direct expression in COS-7 cells, and nucleotide sequence analysis. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids. Expression of high levels of human TNF in Escherichia coli was accomplished under control of the bacteriophage lambda PL promoter and gene N ribosome binding site.     

The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase

A complementary DNA (cDNA) for ubiquitin carboxyl-terminal hydrolase isozyme L3 was cloned from human B cells. The cDNA encodes a protein of 230 amino acids with a molecular mass of 26.182 daltons. The human protein is very similar to the bovine homolog, with only three amino acids differing in over 100 residues compared. The amino acid sequence deduced from the cDNA was 54% identical to that of the neuron-specific protein PGP 9.5. Purification of bovine PGP 9.5 confirmed that it is also a ubiquitin carboxyl-terminal hydrolase. These results suggest that a family of such related proteins exists and that their expression is tissue-specific.     

A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions

Complementary DNA and genomic clones were isolated and sequenced corresponding to rat and human synaptophysin (p38), a major integral membrane protein of synaptic vesicles. The deduced amino acid sequences indicate an evolutionarily highly conserved protein that spans the membrane four times. Both amino and carboxyl termini face the cytoplasm, with the latter containing ten copies of a tyrosine-rich pentapeptide repeat. The structure of synaptophysin suggests that the protein may function as a channel in the synaptic vesicle membrane, with the carboxyl terminus serving as a binding site for cellular factors.     

Sequence of the immunodominant epitope for the surface protein on sporozoites of Plasmodium vivax

Plasmodium vivax is one of the four malaria parasites that cause disease in humans. The structure of the immunodominant repeating peptide of the circumsporozoite (CS) protein of P. vivax was determined. A fragment of P. vivax DNA that encodes this tandemly repeating epitope was isolated by use of an oligonucleotide probe whose sequence is thought to be conserved in CS protein genes. DNA sequence analysis of the P. vivax clone indicates that the CS repeat is nine amino acids in length (Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala). The structure of the repeating region was confirmed with synthetic peptides and monoclonal antibodies directed against P. vivax sporozoites. This information should allow synthesis of a vaccine for P. vivax that is similar to the one being tested for P. falciparum.     

Three-dimensional structures of the TAFII-containing complexes TFIID and TFTC

TBP (TATA-binding protein)-associated factors (TAF(II)s) are components of large multiprotein complexes such as TFIID, TFTC, STAGA, PCAF/GCN5, and SAGA, which play a key role in the regulation of gene expression by RNA polymerase II. The structures of TFIID and TFTC have been determined at 3.5-nanometer resolution by electron microscopy and digital image analysis of single particles. Human TFIID resembles a macromolecular clamp that contains four globular domains organized around a solvent-accessible groove of a size suitable to bind DNA. TFTC is larger and contains five domains, four of which are similar to TFIID.     

Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease

Four clones were isolated from an adult human brain complementary DNA library with an oligonucleotide probe corresponding to the first 20 amino acids of the beta peptide of brain amyloid from Alzheimer's disease. The open reading frame of the sequenced clone coded for 97 amino acids, including the known amino acid sequence of this polypeptide. The 3.5-kilobase messenger RNA was detected in mammalian brains and human thymus. The gene is highly conserved in evolution and has been mapped to human chromosome 21.     

Human lipoprotein lipase complementary DNA sequence

Lipoprotein lipase is a key enzyme of lipid metabolism that acts to hydrolyze triglycerides, providing free fatty acids for cells and affecting the maturation of circulating lipoproteins. It has been proposed that the enzyme plays a role in the development of obesity and atherosclerosis. The human enzyme has been difficult to purify and its protein sequence was heretofore undetermined. A complementary DNA for human lipoprotein lipase that codes for a mature protein of 448 amino acids has now been cloned and sequenced. Analysis of the sequence indicates that human lipoprotein lipase, hepatic lipase, and pancreatic lipase are members of a gene family. Two distinct species of lipoprotein lipase messenger RNA that arise from alternative sites of 3'-terminal polyadenylation were detected in several different tissues.     

Cloning and expression of the human interleukin-6 (BSF-2/IFN beta 2) receptor

Interleukin-6 (IL-6/BSF-2/IFN beta 2) is a multifunctional cytokine that regulates the growth and differentiation of various tissues, and is known particularly for its role in the immune response and acute phase reactions. A complementary DNA encoding the human IL-6 receptor (IL-6-R) has now been isolated. The IL-6-R consists of 468 amino acids, including a signal peptide of approximately 19 amino acids and a domain of approximately 90 amino acids that is similar to a domain in the immunoglobulin (Ig) superfamily. The cytoplasmic domain of approximately 82 amino acids lacks a tyrosine/kinase domain, unlike other growth factor receptors.     

Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart

Neuronal cells express a pp60c-src variant that displays an altered electrophoretic mobility and a different V8 peptide pattern relative to pp60c-src expressed in tissues of non-neuronal origin. To determine whether the neuronal form of pp60c-src is encoded by a brain-specific messenger RNA, a mouse brain complementary DNA (cDNA) library was screened with a chicken c-src probe and a 3.8-kilobase c-src cDNA clone was isolated. This clone encodes a 60-kilodalton protein that differs from chicken or human pp60c-src primarily in having six extra amino acids (Arg-Lys-Val-Asp-Val-Arg) within the NH2-terminal 16 kilodaltons of the molecule. S1 nuclease protection analysis confirmed that brain c-src RNA contains an 18-nucleotide insertion at the position of the extra six amino acids. This insertion occurs at a position that corresponds to a splice junction in the chicken and human c-src genes. The isolated c-src cDNA clone encodes a protein that displays an identical V8 peptide pattern to that observed in pp60c-src isolated from tissues of neuronal origin.     

The structure of rat preproatrial natriuretic factor as defined by a complementary DNA clone

The structure of rat preproatrial natriuretic factor ( preproANF ) was determined by nucleotide sequence analysis of an ANF complementary DNA clone. PreproANF is composed of a hydrophobic leader segment (20 amino acids), a precursor containing one glycosylation site (106 amino acids), and ANF (24 amino acids). Atrial natriuretic factor is located at the carboxyl terminus of the precursor molecule. The human, mouse, and rat genomes each contain a single ANF gene which is highly conserved.     

The Structure and Sequence Analysis of TLR4 Gene in Cattle

Toll-like receptor 4 （TLR4） is essential for initiating the innate response to lipopolysaccharide （LPS） from Gram-negative bacteria by acting as a signal transducting receptor. In order to help in investigating TLR4 as a candidate disease-resistance gene in cows, we isolated the cDNA （GenBank accession no. DQ839566） by RT-PCR and rapid amplification of cDNA ends （RACE） experiments and analyzed the sequence characters by bioinformatics. The results showed that cattle TLR4 gene about 3 739 bp contains an open reading frame of 2 526 bp encoded 841 amino acids （aa）, 470 bp 5′ untranslated region （UTR）, and 743 bp 3′ UTR. Tissue expression profile by RT-PCR indicated that TLR4 gene expresses in mammary glands, liver, muscle, duodenum, fats, uterus, kidneys, hearts, lungs, pancreas, and ovary. TLR4 protein domain predicted by bioinformatics consists of signal peptide, transmembrane helices domain, 3 sorts of leucine-rich repeat domains （LRR, LRR-TYP, and LRRCT）, and a toll-interleukinl-resistance domain （TIR）. Leucine-rich repeat domains were related with recognizing a broad of pathogen-associated molecular patterns （PAMP） from pathogen, and TIR domain for downstream signaling transduction was most conservative （98% identify） than other domains after alignment of protein from ovine, porcine, human, and mouse. In addition, a 470 bp 5′-flanking region sequence was amplified by PCR, and 15 putative DNA binding sites were predicted, but this sequence lacks TATA box, CCAAT character, and GC-rich regions.     

Aldolase activity of a Plasmodium falciparum protein with protective properties

Immunization with a 41-kilodalton blood stage antigen (p41) of Plasmodium falciparum induces immunity to malaria in monkeys. However, antigenic polymorphism and repetitive amino acids commonly found in protective antigens complicate vaccine development. The gene encoding p41 has now been cloned and analyzed. Sequencing and hybridization studies revealed that the gene structure is highly conserved in 14 parasite isolates from three continents. This finding and the lack of repetitive amino acids in the translated DNA sequence may indicate that p41 has an essential function. In this study the protein was found to be 60 percent homologous to the key glycolytic enzyme aldolase from vertebrates, and the affinity-purified p41 protein from parasites showed aldolase activity.     

Molecular cloning and expression of a human B-cell growth factor gene in Escherichia coli

A human B-cell growth factor (BCGF) (12 kilodaltons) supports the clonal proliferation of B lymphocytes. A clone was isolated that contained the proper structural sequence to encode biologically active, 12-kilodalton BCGF in Escherichia coli and to hybridize to a specific messenger RNA, identified by in vitro translation in Xenopus laevis oocytes. A relatively hydrophobic region of 18 amino acids was found at the amino terminal of the 124-amino acid-long polypeptide. The carboxyl terminal is composed of at least 32 amino acids that are derived from nucleotide sequences bearing significant homology to the Alu repeat family.     

海岛棉DREB基因的克隆及植物表达载体的构建

根据GenBank上公布的棉花GhDREB1基因序列设计特异性引物,从海岛棉中克隆了一个与DREB类基因同源的cDNA序列,命名为XHDREB。序列分析表明,该cDNA序列长度为651 bp,推测含有216个氨基酸,分子量为24.6 kDa,等电点为9.46。同源性分析表明,它与GhDREB1的氨基酸序列同源性为85.19%。所编码蛋白具备DREB1转录因子的特征:含有58个氨基酸组成的AP2 DNA结合域以及两个DREB1/CBF特征氨基酸序列PKKRAGRKKFRETR和DSAWR。然后将XHDREB基因连接到pCAMBIA2300-35SOCS质粒上,构建了植物表达载体。