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. In this study we used microarrays to analyse the influence of BvgA/S or modulation on the expression of nearly 200 selected genes. With the exception of one vrg, all previously known vags and vrgs were correctly assigned as such, and the microarray analyses identified several new vags and vrgs, including genes coding for putative autotransporters, two-component systems, extracellular sigma factors, the adenylate cyclase accessory genes cyaBDE, and two genes coding for components of a type III secretion system. For most of the new vrgs and vags the results of the microarray analyses were confirmed by RT-PCR analysis and/or lacZfusions. The degree of regulation and modulation varied between genes, and showed a continuum from strongly BvgA/S-activated genes to strongly BvgA/S-repressed genes. The microarray analyses also led to the identification of a subset of vags and vrgs that are differentially regulated and modulated by MgSO(4) or nicotinic acid, indicating that these genes may be targets for multiple regulatory circuits. For example, the expression of bilA, a gene predicted to encode an intimin-like protein, was found to be activated by BvgA/S and up-modulated by nicotinic acid. Furthermore, surprisingly, in the strain analysed here, which produces only type 2 fimbriae, the fim3 gene was identified as a vrg, while fim2 was confirmed to be a vag.

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. A panel of open-reading frames (ORFs) from the C. pneumoniae genome sequence was screened for ability to elicit protective responses. Balb/c mice immunized with DNA containing the ORFs were tested for their ability to limit lung infection following an intranasal challenge. Immunization with DNA encoding the major outer membrane protein or an ADP/ATP translocase (Npt1(Cp)) of C. pneumoniae resulted in a reduced bacteria load in the lung after challenge. The identification of these antigens as protective is a significant step toward development of a C. pneumoniae vaccine and demonstrates the feasibility of using a DNA immunization strategy to screen the C. pneumoniae genome for other protective ORFs.

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. Under optimal conditions as analyzed by FACS, up to 60% of Cos-7 and 50% of CHO cells can be transfected. Furthermore, a comparison of different lipids (Transfectam, RPR120535b, TC1-12 and GAP-DLRIE/DOPE) suggests that lipids with multiple amine groups are more efficient for the transfection of filamentous phage.

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. To overcome this problem two complementary strategies have been used: a nonspecific polyclonal activation of B cells, and a secondary PCR amplification technique to ensure recovery of Ig messengers in large amount and without introducing any bias.

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.

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. The heat-inducible expression of the Int recombinase allows perfect control of recombination. Selection of the recombinant phagemid is made possible by the assembly, in vivo, of a genetic marker (chloramphenicol resistance) created only after the correct recombination event. Theoretically, all possible associations between the VL and VH sequences should be obtained, and it should be possible to generate multicombinatorial libraries of close to 10(12) clones.

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. There are 17 putative binding sites for heat-shock transcription factor (HSF), including three clusters of multiple binding sites. We show that this upstream region is sufficient to direct heat-inducible expression of a hsp70A1::cat hybrid gene in mouse and human cells.

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. An endpoint limiting dilution assay was used to quantify cell survival to lethal stresses. Two test plasmacytoma cell lines (C1.18.1 and J558) and an hsp68-positive myeloma, XC1.1/51, used as a control, were examined. All showed recovery when pretreated for 10 min at 44 degrees C 2 h before exposure to otherwise lethal stresses of 1 to 4 h at 43 degrees C. Similar results were obtained with the Friend erythroleukemia line D1B, which we have also shown not to express hsp68. These results indicate that hsp68 is not required for protection against thermal stresses in mouse cells.

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. However, the synthesis of both hsp 89 and hsp 70 was enhanced. Northern blot analysis of cytoplasmic RNA extracted from control and stressed cells indicated that hsp 68 mRNA was absent, even after stresses of up to 1 h at 45 degrees C. Differentiation induced by dimethyl sulphoxide (DMSO) (monitored by the induction of globin synthesis) had no effect on hsp 68 expression in D1B cells; also, hsp 68 could not be induced at various stages of differentiation (0-72 h). Southern blot analysis showed that all three hsp-68 genes were present and not rearranged, and apparently did not carry any deletion in their 5' ends. To determine whether methylation could be involved in maintaining the genes in their silent state, we treated cells with 10 microM 5-azacytidine for 48 h. No hsp 68 expression was observed following such treatment in either undifferentiated or DMSO-induced differentiated D1B cells. Furthermore, Southern blot analysis of MspI/HpaII-digested genomic D1B DNA did not display any differences in methylation patterns around the promoter region of the probed gene compared with control cells, indicating that methylation is not involved in hsp-68 repression. When chimeric plasmids carrying the bacterial chloramphenicol acetyl transferase gene under regulation of the mouse hsp-68 or Drosophila hsp-70 promoters were transfected into D1B cells, minimal (2-fold) or no induction was observed, in contrast with the 60-fold induction seen in a control myeloma cell line. These results suggest a trans-acting mechanism of hsp-68 repression in erythroleukemia cells.

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. Cell lines examined included a T lymphoma, a pre-B lymphocyte, and a non-B-non-T tumor cell line, as well as an Ig-nonproducing myeloma of undetermined differentiated status. Nonexpression is strain-independent as observed in BALB/c and C3H plasmacytomas. Based on S1 nuclease analysis using a cloned genomic hsp-68 probe, nonexpression is caused by the absence of hsp-68 mRNA following heat shock. A time-course experiment suggests that rapid degradation of mRNA does not occur, implying that the block is most likely at the transcriptional level. Southern blot analysis does not indicate any minor deletions around the region of transcription initiation, at least in the probed hsp-68 gene. These results suggest that the absence of hsp-68 gene expression may be a reflection of the differentiated and (or) transformed state of murine plasma cells, possibly through the absence or deregulation of a regulatory factor required for induction of heat-shock 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 (4.1%) following differentiation. The alpha-1 globin genes were heavily methylated at HpaII sites in EC cells and in differentiated derivatives of these EC cells. The MEL cells had only 3.3% of their genomic deoxycytidine methylated and no significant change occurred following differentiation. The alpha-1 globin genes of MEL cells were much less methylated than the same genes in EC cells and no change in this methylation pattern accompanied the induction of hemoglobin synthesis. In EC X MEL cell hybrids which express the alpha-1 globin genes from both parents, the EC-derived genes had become demethylated. These results are consistent with the general model that DNA hypomethylation is correlated with expression of that DNA and that gene activation is accompanied by DNA demethylation. We have also measured the 5MC content of DNA isolated from nuclease-treated EC nuclei. Unexpectedly, the DNase I sensitive chromatin contained a large proportion of 5MC. This result, along with the work of others, suggests that nuclease sensitivity may often reflect the transcriptional activity of chromatin in somatic cells, but is not indicative of the active state in pluripotent EC cells.

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. The unique mitochondrial asparaginyl- and prolyl-tRNAs, not previously identified in mammalian cells, were shown to hybridize to mtDNA. Mitochondrial leucyl-tRNA separated into 3 peaks on RPC-5 and the first species was shown to be different than a combination of the other two by molecular size and partial RNase T1 digestion patterns. Each was coded by a separate gene on mtDNA as shown by partial additivity of hybridization. Separate genes for mitochondrial tRNAMetm and tRNAMetf, separated by RPC-5 chromatography, were also demonstrated. These results bring to 21 the number of individual tRNAs coded by mammalian mtDNA.

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. In each case, the number and electrophoretic mobility of the proteins was the same as mitochondrially synthesized proteins of CHO TK- cells labelled with [35S]methionine. A tRNAAsn could be charged in isolated CHO TK- cell mitochondria and the asparaginyl-tRNA was found to elute before its cytosolic counterpart on an RPC-5 column and to have a higher mobility on polyacrylamide slab gels run under denaturing conditions. This is the first demonstration of a unique mitochondrial asparaginyl-tRNA.

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. Mitochondrial leucyl-tRNAs sedimented more slowly than their cytosolic counterparts, again indicating a lower molecular weight. The apparent molecular weights of the mitochondrial methionyl-tRNAs were identical or only slightly lower than their cytosolic counterparts as determined by polyacrylamide slab gel electrophoresis but both mitochondrial methionyl-tRNA and formylmethionyl-tRNA sedimented slightly more slowly than cytolsolic methionyl-tRNA. It is suggested that mitochondrial tRNAs fall into the size range of other t RNAs and might be uniform in size.

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. Cystolic tRNALeu was not cleaved. These results are discussed in relationship to the reported low content of methylated nucleosides in mitochondrial 4 S RNA.

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. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.