DNA (Mary Ann Liebert, Inc.)最新文献

A new vector, CDM8PI, has been constructed. It was derived from the plasmid expression vector CDM8, which has been used in the epitope-loss mutant isolation technique to map the epitopes on cell-surface proteins. The new vector allows the production of fusion proteins between normally secreted proteins and the membrane anchor moiety from a cell-surface protein, LFA-3, thereby expressing the fusion proteins on the cell surface. The vector extends the application of the epitope-loss mutant isolation technique to secreted proteins. The vector also allows the easy recovery of mutated proteins in unfused forms after the immunoselection and characterization.

I describe a general and rapid procedure allowing the isolation of specialized lambda transducing phages from a lambda library by lysogenic complementation of defective mutants of Escherichia coli. As an example, the cloning of the E. coli fabE gene and of two other adjacent genetic determinants is presented. Subcloning and determination of its nucleotide sequence reveals that fabE codes for the biotin carboxyl carrier protein (BCCP), one of the three subunits of acetyl coenzyme A carboxylase.

185c-neu is a member of a family of growth factor receptors with tyrosine kinase activity. A point mutation in the transmembrane region leads to activation of the enzymatic domain. We demonstrate that TPA (phorbol-12-myristate-13-acetate) stimulates the phosphorylation of p185c-neu on serine and threonine residues coincident with the inhibition of its intrinsic tyrosine kinase and the proliferation of cells that express it. The tyrosine kinase activity as well as the phosphorylation pattern of serine and threonine residues of oncogenic p185 (p185neu) and the growth of p185neu-expressing cells are not influenced by TPA. These observations indicate that the functional activity of p185c-neu can be regulated through protein kinase C (PKC) but the transmembrane point mutation present in p185neu renders it refractory to serine/threonine kinase regulation.

We cloned and determined the DNA sequence of a novel gene (termed RD or D6S45) situated in the class III region of the human major histocompatibility complex (HLA) between the Bf and C4A complement genes. The coding region of the gene is contained in 10 exons spread over approximately 6 kb of DNA. The encoded protein is predicted to contain 371 amino acid residues with a molecular weight of 41,000. The predicted amino acid sequence is notable for a central region containing 24 consecutive pairs of alternating basic (Arg) and acidic (Asp or Glu) residues similar to, but more strictly alternating than, those seen in the 70K protein of the U1 small nuclear ribonucleoprotein (snRNP). There is a high degree of homology both at the amino acid and DNA level between the human RD gene and its murine homology. Although the central region is highly repetitious and contains a high proportion (13%) of CpG dinucleotides-both features that might predispose to frequent mutations-sequence analysis of this region in genes amplified from six individuals revealed no polymorphism.

Different results are obtained when DNA in aqueous solution and DNA in biological tissue are exposed to ultrasound. At intensities of ultrasound comparable to those applied clinically, ultrasonication is able to degrade purified DNA in aqueous solution, making ultrasonication a useful tool for preparing DNA fragments in vitro. Ultrasonic degradation of DNA in solution occurs by breaking hydrogen bonds and by single-strand and double-strand ruptures of the DNA helix. Two mechanisms are mainly responsible: cavitation and a thermal or mechanical effect. Stable cavitation is seen at low intensities of ultrasound. Increasing the intensity of the ultrasound above 2 W/cm2 is followed by increases in single-strand ruptures due to the creation of free radicals by transient cavitation. Following sonication, the distribution of the resulting DNA fragments approaches a lower size limit of 100-500 bp. Breaks in the DNA helix occur mainly between oxygen and carbon atoms, resulting in DNA fragments with a phosphorylated 5' end and a free alcohol at the 3' end. The relative lack of specificity in degrading the DNA helix makes ultrasonication a complementary alternative to the highly specific fragmentation obtained by restriction endonucleases.

The complete mRNA sequence of the human liver-type phosphofructokinase (hPFKL) was determined. The sequence included 55 nucleotides of 5' and 515 of 3' noncoding regions, as well as 2,337 nucleotides encoding the 779 amino acids of the hPFKL. Extensive similarity (approximately 90%) in the coding region was observed between the hPFKL and the mouse PFKL, whereas the degree of similarity between different types of PFK, i.e., hPFKL and human muscle-type PFK (hPFKM), was merely 68%. Nevertheless, striking similarity between these different types of PFK was noticed when the amino acid residues creating the various active sites of the enzyme were compared. Human PFK L- and M-specific probes were constructed and used to quantitate the mRNA levels in fetal and adult brains and fetal liver. It was found that while relative amount of PFKL mRNA in adult brain was one-fourth of that detected in fetal brain the level of PFKM mRNA in adult brain was slightly higher than in fetal tissue, suggesting that PFK expression might be controlled at the transcriptional level.

We have identified and cloned human cDNA for the Ca2+-binding subunit of calcineurin, the brain isozyme of the Ca2+/calmodulin-stimulated protein phosphatase. The 2.5-kb cDNA has an open reading frame of 510 bp, a leader sequence of at least 500 bp, and a 1,277-bp 3'-noncoding sequence. The deduced sequence of the human protein differs from bovine brain calcineurin B by an additional valine at the carboxyl terminus and substitution of Met-11 and Ser-153 by cysteine. A partial clone of the mouse protein corresponding to amino acids 75-150 was also isolated. This portion of the human and mouse protein sequence is identical, with the DNA sequences showing 94% identity. The respective mRNAs in human and mouse are also of similar size. As was observed with protein levels, mRNA abundance in brain is 20-60 times that found in other tissues with the exception of HeLa cells which, like brain, contain abundant calcineurin B mRNA.

Even though the rev gene of the human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, high levels of rev also downregulate viral gene expression. As the degree of rev protein expression exceeds expression of wild-type virus, a gradient of decreasing viral mRNA synthesis becomes evident. The target sequence for this downregulation resides outside of trans-activating region (TAR) and upstream from the enhancer sequences in the long terminal repeat (LTR), suggesting that regulation is at a transcriptional level.

Manoil and Beckwith (1985) have constructed a transposon, TnphoA, that permits the generation of hybrid proteins composed of alkaline phosphatase (AP) lacking its signal peptide fused to amino-terminal sequences of other proteins. This transposon has been used to localize export signals and analyze membrane topology of bacterial proteins. We have applied this approach to the membrane fusion protein (F) of respiratory syncytial virus (RSV). The transposon TnphoA and a plasmid directing bacterial expression of the F gene were used to construct F-AP hybrids. These hybrids yielded AP activity, indicating the presence of viral sequences that promoted protein transport through the cytoplasmic membrane. Sequence analysis showed that TnphoA was inserted at four different positions within the F1 subunit. Deletion of the hydrophobic F1 amino-terminus (fusion-related domain) resulted in AP transport to the periplasm, suggesting that the hydrophobic amino-terminus of the F2 subunit is sufficient to promote protein export. Some hybrids were apparently cleaved at or near the F2/F1 junction. The periplasmic localization of an uncleaved hybrid strongly suggested that the fusion-related domain of the F protein, when in the uncleaved F0 precursor, can be moved across the bacterial cytoplasmic membrane. Although these results apply to the recombinant F protein, they agree with the presumed signal sequence and membrane topology of the native F glycoprotein. Thus, this method may be useful in determining membrane topology and in localizing important domains of viral proteins.

We describe a novel application of the polymerase chain reaction (PCR) technique of DNA amplification to study the exon-intron structure of the rat plasminogen activator inhibitor (PAI-1) gene. This technique is relatively simple and also allows the isolation of introns for sequencing. Primers were selected based on a knowledge of the cDNA sequences of human and rat PAI-1 and of the gene structure of human PAI-1. However, knowledge of a cDNA sequence and/or the structure of a gene in another species is not a prerequisite. Sequences selected from positions along the cDNA of interest could be used to amplify the DNA either from an isolated but uncharacterized gene or directly from genomic DNA, making this technique generally applicable. Thus, this method is a useful advance in the study of gene structure and evolution.