Differential modulation of Bordetella pertussis virulence genes as evidenced by DNA microarray analysis.

The production of most factors involved in Bordetella pertussis virulence is controlled by a two-component regulatory system termed BvgA/S. In the Bvg+ phase virulence-activated genes (vags) are expressed, and virulence-repressed genes (vrgs) are down-regulated. The expression of these genes can also be modulated by MgSO(4) or nicotinic acid.

Cloning of interleukin-4 delta2 splice variant (IL-4delta2) in chimpanzee and cynomolgus macaque: phylogenetic analysis of delta2 splice variant appearance, and implications for the study of IL-4-driven immune processes.

The human interleukin-4 ( IL-4) gene produces an exon 2-lacking alternative splice variant, termed IL-4delta2, and described as a naturally occurring antagonist of IL-4-driven activity. We report the isolation of an IL-4delta2 cDNA from chimpanzee ( Pan troglodytes) bone marrow samples and cynomolgus macaque ( Macaca fascicularis) activated peripheral lymph node cells. The complete IL-4 cDNA sequence from chimpanzee is also provided for the first time.

Experimental design optimization of filamentous phage transfection into mammalian cells by cationic lipids.

A previous study showed that filamentous phage could be efficiently transfected into mammalian cells in the presence of the cationic lipid Transfectam. In the present study, we used an experimental plan based on a uniform network (Doehlert) matrix to estimate optimal transfection conditions in two different cell lines, CHO and Cos-7. Using the cationic lipid RPR120535b as a model, we show that optimal conditions can be determined much more readily than with standard response curves.

Use of a mouse lung challenge model to identify antigens protective against Chlamydia pneumoniae lung infection.

Chlamydia pneumoniae is emerging as a significant human pathogen. Infection causes a range of respiratory tract diseases and is associated with atherosclerosis. A vaccine could provide a considerable public health benefit; however, antigens able to elicit a protective immune response are largely unknown.

High affinity human antibodies by phage display.

The development of phage display has now made it possible to consider the isolation of human antibodies directly without immunization. Recent advances in the field of human immunogenetics and in phage technology have led to the assembly of 'naive' human repertoires in vitro whose complexity approach that of the natural immune system. Screening of these libraries has allowed the isolation in one step of antibodies with affinities in the nanomolar range.

Full-scale 'naïve' human antibody repertoires assembled from VH and VL variable regions.

Very large 'naïve' human antibody repertoires have been obtained from RT-PCR cloned VH and VL variable regions. They are used as starting material for the assembly of medium sized combinatorial libraries or so called multicombinatorial libraries. In nonimmunized individuals immunoglobulin messenger RNAs are poorly expressed, which can be a serious limitation for cloning efficiency.

A new phage display system to construct multicombinatorial libraries of very large antibody repertoires.

We present an easy and efficient technique for the construction of large phage-displayed antibody (Ab) repertoires through the recombination of two separate heavy (VH) and light (VL) chain gene libraries. Here, the system has been applied to the display of a chimpanzee anti-HIV gp160 Ab. The process, which makes use of lambda phage att recombination sites, leads to the irreversible physical association between plasmid and phagemid vectors carrying, respectively, VL and VH sequences.

Structure and expression of an inducible HSP70-encoding gene from Mus musculus.

We have determined the nucleotide sequence of a stress-inducible mouse Hsp70-encoding gene named hsp70A1. The gene encodes a 641-amino-acid protein whose deduced sequence is similar to those of other members of the HSP70 family. The 5' end (tsp) of a heat-inducible mRNA is 225 bp upstream from the start codon, and several consensus recognition sequences for transcription factors lie upstream from this tsp.

The major inducible heat shock protein hsp68 is not required for acquisition of thermal resistance in mouse plasmacytoma cell lines.

In mouse cells, the major inducible heat shock protein is a protein of 68,000 daltons (hsp68). We have previously shown that mouse plasmacytomas do not express hsp68. We have now made use of these natural mutants to assess the contribution of hsp68 to acquired thermotolerance.

The major heat-shock protein hsp 68 is not induced by stress in mouse erythroleukemia cell lines.

Most mammalian cells respond to brief incubation at elevated temperatures by enhanced or new synthesis of a set of heat-shock proteins (hsp). In mouse cells, as determined by SDS--one-dimensional gel electrophoresis, the most prominent hsps have molecular masses of approximately 89,000, 70,000, and 68,000 Da. When the heat-shock response of the mouse erythroleukemia cell line D1B, or two other DBA/2 cell lines (707C1 and 745C2), was examined by [35S]methionine labelling, following heat shocks of 10 min at 42 or 44 degrees C, or 1 h at 45 degrees C, no protein band corresponding to hsp 68 was observed.

Murine plasmacytomas constitute a class of natural heat-shock variants in which the major inducible hsp-68 gene is not expressed.

Analysis of the heat-shock response in murine plasmacytomas reveals that, as demonstrated previously for the MPC-11 cell line, the genes coding for the 68-kilodalton heat-shock protein (hsp-68) are not expressed upon heat shock or sodium arsenite treatment. Noninduction is unique to the normally coordinated set of three hsp-68 genes since at least two other heat-shock protein genes (hsp-70 and hsp-89) are properly induced. No other lymphoid cell line was found to possess silent hsp-68 genes.

Nonexpression of a major heat shock gene in mouse plasmacytoma MPC-11.

The major heat shock gene coding for a 68,000-dalton protein was found to be silent in mouse plasmacytoma MPC-11 in both control and stressed cells. The block appears to be at the level of transcription, although RNA processing or instability has not been ruled out. It is not caused by a major deletion or rearrangement of the gene.

DNA methylation in differentiating mouse teratocarcinoma and erythroleukemia cells.

We have measured the content of 5-methylcytosine (5MC) in the genomic DNA of differentiated and undifferentiated cultures of murine embryonal carcinoma (EC) and murine erythroleukemia (MEL) cells. A large proportion of deoxycytosine residues were methylated in EC cells (4.6%) and this proportion dropped significantly (4.

Mammalian mitochondrial transfer RNAs: chromatographic properties, size and origin.

Incubation of isolated rat liver mitochondria with radioactive amino acids resulted in the charging of tRNAs for arginine, asparagine, leucine, lysine, methionine, proline and valine. The aminoacyl-tRNAs were shown to be distinct from their cytosolic counterparts by chromatography on RPC-5. By electrophoresis on urea polyacrylamide slab gels it was found that all these mitochondrial aminoacyl-tRNAs were about 70-76 nucleotides long.

Biochemical and genetic approaches to the study of mammalian mitochondrial tRNAs.

The possible existence of mammalian mitochondrial asparaginyl-tRNA has been examined using a variety of approaches. [3H]Asparagine was incorporated into protein by mitochondria of the Chinese hamster ovary (CHO) cell line Asn-7, which has a temperature-sensitive cytosolic asparaginyl-tRNA synthetase, either in the presence of cycloheximide or at a nonpermissive temperature. Isolated mitochondria of CHO thymidine kinase minus (TK-) cells also incorporated the amino acid into protein.

Chemical and physical properties of mammalian mitochondrial aminoacyl-transfer RNAs. II. Analysis of 7-methylguanosine in mitochondrial and cytosolic aminoacyl-transfer RNAs.

The 7-methylguanosine (m7G) content of two individual mitochondrial tRNAs, labelled in the aminoacyl moiety was assayed by the specific cleavage of the tRNA at this nucleotide followed by electrophoretic analysis to identify the 3'-terminal fragment of the tRNA. Neither Syriam hamster mitochondrial tRNALeu nor tRNAMet were found to contain m7G. In contrast, cytosolic tRNAMetS were cleaved indicating the presence of m7G, apparently 27--28 and 29 nucleotides from their 3' terminus.

Chemical and physical properties of mammalian mitochondrial aminoacyl-transfer RNAs. I. Molecular weights of mitochondrial leucyl- and methionyl-transfer RNAs.

The sedimentation and electrophoretic properties of Syrian hamster cytosolix and mitochondrial methionyl- and leucyl- +RNAs have been compared under denaturing conditions. Mitochondrial leucyl-tRNA could be separated into three species by chromatography on RPC-5. Their apparent molecule weights as determined by polyacrylamide slab gel elecltrophoresis were 23 000 for one species and 24 000 for the other two compared to the five cytosolic leucyl-tRNA species whose apparent molecular weights ranged from 26 000 to 28 000.

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE.