Direct resorption of bone by human monocytes.

Cultured human peripheral blood monocytes stimulate the release of bone mineral and matrix from killed long bones of fetal rats. These effects were inhibited by cortisol but were not altered by hormones that normally stimulate osteoclastic bone resorption. There was no evidence of morphologic differentiation of the monocytes into osteoclasts during bone resorption.

Antibody arrays for high-throughput screening of antibody-antigen interactions.

We have developed a novel technique for high-throughput screening of recombinant antibodies, based on the creation of antibody arrays. Our method uses robotic picking and high-density gridding of bacteria containing antibody genes followed by filter-based enzyme-linked immunosorbent assay (ELISA) screening to identify clones that express binding antibody fragments. By eliminating the need for liquid handling, we can thereby screen up to 18,342 different antibody clones at a time and, because the clones are arrayed from master stocks, the same antibodies can be double spotted and screened simultaneously against 15 different antigens. We have used our technique in several different applications, including isolating antibodies against impure proteins and complex antigens, where several rounds of phage display often fail. Our results indicate that antibody arrays can be used to identify differentially expressed proteins.

A simple and efficient method for the isolation of differentially expressed genes.

A simple and reproducible general approach for the isolation of differentially expressed genes is described. Digestion of cDNAs with a class IIs restriction endonuclease produces fragments with every combination of possible bases in the cohesive ends. Under stringent conditions, the specific ligation of adaptors with perfectly complementary overhangs partitions the cDNA fragments into non-overlapping subpopulations. Internal cDNA restriction fragments are exponentially amplified by adaptor primer PCR and visualised by non-denaturing polyacrylamide gel electrophoresis. The power of the technology was demonstrated using a rat model of pressure-induced left-ventricular hypertrophy (LVH). A set of 29 fragments, derived from a sample (6 %) of the possible adaptor pool combinations, displayed apparent differential expression. The differential expression of 19 (66 %) were confirmed by Northern blot analysis. Sequence analysis identified both genes known to be upregulated in LVH, and novel genes. The fidelity of adaptor ligation was demonstrated by the isolation of known gene fragments by appropriate adaptor combinations. The spiking of mRNA populations with known amounts of a synthetic mRNA demonstrated a current sensitivity equivalent to the detection of transcripts expressed at the level of as little as 1 in 10,000 molecules.

Agents that cause a high frequency of genetic change from [psi+] to [psi-] in Saccharomyces cerevisiae.

The [psi] factor of yeast is cytoplasmically inherited. Singh, Helms and Sherman (1979) reported that high concentrations of KCl and of ethylene glycol induce the genetic change from [psi+] to [psi-]. In this study, the following agents have been shown to induce the same genetic change: guanidine hydrochloride at 1 mM, dimethyl sulfoxide at 2.5% v/v and ethanol or methanol at 10% v/v. It is likely that a number of other agents also cause the change, namely 2 M glycerol, M succinate, M glutamate and M MgCl2. Most of these agents induce the change at very high frequencies; with some, the frequency is 100%. Although the observed phenotypic change can also occur as a result of chromosomal gene mutation, no changes of this type were identified. Some of the agents also cause mutation from [rho+] to [rho-] and from killer to sensitive.

Identification of potential stroke targets by lentiviral vector mediated overexpression of HIF-1 alpha and HIF-2 alpha in a primary neuronal model of hypoxia.

The identification of genes differentially regulated by ischemia will lead to an improved understanding of cell death pathways such as those involved in the neuronal loss observed following a stroke. Furthermore, the characterization of such pathways could facilitate the identification of novel targets for stroke therapy. We have used a novel approach to amplify differential gene expression patterns in a primary neuronal model of stroke by employing a lentiviral vector system to specifically bias the transcriptional activation of hypoxically regulated genes. Overexpression of the hypoxia-induced transcription factor subunits HIF-1 alpha and HIF-2 alpha elevated hypoxia-mediated transcription of many known HIF-regulated genes well above control levels. Furthermore, many potentially novel HIF-regulated genes were discovered that were not previously identified as hypoxically regulated. Most of the novel genes identified were activated by a combination of HIF-2 alpha overexpression and hypoxic insult. These included several genes with particular importance in cell survival pathways and of potential therapeutic value. Hypoxic induction of HIF-2 alpha may therefore be a critical factor in mediating protective responses against ischemic injury. Further investigation of the genes identified in this study may provide increased understanding of the neuronal response to hypoxia and may uncover novel therapeutic targets for the treatment of cerebral ischemia.

Reference cDNA library facilities available from European sources.

cDNA libraries are the cornerstone of efforts to identify the relatively small regions of genomes that are responsible for biological effects. Gene hunter seeking candidate genes, via a variety of approaches, ultimately focus on the cloning, sequencing, and expression of cDNAs. Assistance is now available to researchers in the form of genome programs, whose initial goals include assembly of a complete collection of expressed sequences derived from the genome of interest. The concept of reference sets of cDNA libraries is that the aims of genome programs are served most effectively by different laboratories working on a common set of high-quality arrayed cDNA libraries, using different experimental approaches, thereby reducing unnecessary duplication of effort, and maximizing the amount of information that one set of resources can provide.

Biochemical and immunological characterisation of mutants induced in V79 Chinese hamster cells by a benzo[a]pyrene diolepoxide.

A series of 8-azaguanine resistant mutants was induced by treatment of V79 Chinese hamster cells with either r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (antiBPDE) or methylnitrosourea (MNU). Hypoxanthine phosphoribosyltransferase (HPRT) activity in the mutants was determined for both hypoxanthine and azaguanine as substrates. With antiserum to purified brain HPRT, cross-reacting material was also determined and analysed by two dimensional polyacrylamide gel electrophoresis. By these criteria mutants induced by anti-BPDE or MNU did not differ appreciably and the data obtained was consistent with the induction of point mutations by both carcinogens. The relevance of these results to the correlation of carcinogenicity with mutagenicity in V79 cells, but not in bacteria, is discussed.

Comparative genome sequence analysis of the Bpa/Str region in mouse and Man.

The progress of human and mouse genome sequencing programs presages the possibility of systematic cross-species comparison of the two genomes as a powerful tool for gene and regulatory element identification. As the opportunities to perform comparative sequence analysis emerge, it is important to develop parameters for such analyses and to examine the outcomes of cross-species comparison. Our analysis used gene prediction and a database search of 430 kb of genomic sequence covering the Bpa/Str region of the mouse X chromosome, and 745 kb of genomic sequence from the homologous human X chromosome region. We identified 11 genes in mouse and 13 genes and two pseudogenes in human. In addition, we compared the mouse and human sequences using pairwise alignment and searches for evolutionary conserved regions (ECRs) exceeding a defined threshold of sequence identity. This approach aided the identification of at least four further putative conserved genes in the region. Comparative sequencing revealed that this region is a mosaic in evolutionary terms, with considerably more rearrangement between the two species than realized previously from comparative mapping studies. Surprisingly, this region showed an extremely high LINE and low SINE content, low G+C content, and yet a relatively high gene density, in contrast to the low gene density usually associated with such regions.