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PubMed2004

High throughput determination of gains and losses of genetic material using high resolution BAC arrays and comparative genomic hybridization.

Chromosome analysis has been a cornerstone both for the identification of genetic defects that predispose to a variety of genetic syndromes as well as for the analysis of cancer progression. The relatively low resolution of metaphase chromosomes, however, only allows characterization of major genetic events which are defined at the megabase level. The development of the human genome-wide bacterial artificial chromosome (BACs) libraries which were used as templates for the human genome project made it possible to design microarrays containing these BACs which can theoretically span the genome uninterrupted. Comparative genomic hybridization to these arrays using test and reference DNA samples reveals numerical chromosome abnormalities (deletions, gains and amplifications) which can be accurately defined with a resolution depending on the density of the arrays. Analysis of test DNA samples using these arrays reveals low level deletions and amplifications that cannot be detected by chromosome analysis and provides a global view of these genetic changes in a single overnight hybridization using a high throughput approach. The extent of the genetic changes can then be determined precisely and the gene content of the affected regions established. These BAC arrays have widespread application to the analysis of constitutional genetic abnormalities associated with human diseases as well as cancer patients and their tumors. The development of similar BAC arrays for the mouse genome means that it is now possible to extend the CGHa approach to the study of genetic disorders and cancer models in mice.

Animals↗
PubMed2005

Isolation and structural analysis of efficient autonomously replicating sequences (ARSs) of the yeast Candida utilis.

The industrially important yeast Candida utilis is widely used in production of food and medical materials, but its host-vector system has not been well developed. We screened for compact and efficient ARSs to construct practically useful vectors. The C. utilis strain AHU3053 was found to be efficiently transformed by the conventional lithium acetate method and was used as the host. The C. utilis IAM4264 genomic library was constructed by inserting the partial Sau3AI digests in pRI51, which has a kanMX gene expressible in C. utilis. By examining 98 C. utilis G418-resistant transformants, five plasmids had the highest ARS activity. By trimming of the inserts, the 1490 and 552 bp fragments with transformation activity of over 10(3)/microg DNA were obtained from ARS3 and ARS4, respectively. Although several sequences identical to S. cerevisiae ARS consensus sequences (ACSs) were found in ARS3 and ARS4, our deletion analysis indicated that these were not essential for the activity. Because the minimal functional ARS fragment was also several-fold larger than that of S. cerevisiae, the C. utilis ARSs have some unique characteristics resembling the Sz. pombe ARSs. These ARSs were functional in other C. utilis strains tested and useful for constructing practical vectors.

Base Sequence↗
PubMed1988

Filter-supported preparation of lambda phage DNA.

A rapid and simple method is described for the isolation of DNA from phage lambda which requires neither special equipment nor expensive material such as cesium chloride for ultracentrifugation nor extractions with organic solvents or ethanol precipitation. Microgram quantities of lambda DNA are obtained in less than 2 h from 90-mm plate lysates or 5-ml liquid cultures. The method allows the simultaneous isolation of large numbers of probes, e.g., clones from phage libraries. Lambda phages are precipitated by polyethylene glycol/sodium chloride and recovered by low speed centrifugation onto glass fiber filters positioned in disposable syringes. The DNA of phages is released by a 50% formamide/4 M sodium perchlorate solution, washed in filter-bound form, eluted with a small volume of low-salt buffer or water, and finally recovered by centrifugation. Comparison of the DNA isolated by this method with that obtained by two conventional procedures reveals both a similar recovery and a similar suitability for restriction enzyme digestion and subcloning.

Bacteriophage lambda↗
PubMed1995

Characterization of a Schistosoma mansoni cDNA encoding a B-like cyclophilin and its expression in Escherichia coli.

A cDNA encoding a Schistosoma mansoni cyclophilin (SmCyP) has been cloned by polymerase chain reaction amplification using degenerate oligonucleotides based on known conserved cyclophilin (CyP) sequences and by screening an expression cDNA library. The cDNA sequence encodes a 21.5-kDa protein, which shares 59% sequence identity with human CyP B. The SmCyP protein was expressed in Escherichia coli with a hexahistidine affinity tag at its amino terminus and antibodies to the purified (His6)-SmCyP fusion protein were raised in a rabbit. Fractionation of parasite material followed by immunoblot analysis revealed that schistosome CyP is a soluble protein. The N-terminus of the predicted protein contains a hydrophobic region, suggestive of a signal sequence. Accordingly, a recombinant SmCyP protein, lacking the first 23 amino acids was found to share the same gel electrophoretic mobility as the parasite-derived CyP protein, suggesting cleavage of a leader sequence. Hybridization of genomic DNA to a full-length cDNA probe indicates that the SmCyP gene is present as a single copy. Immunohistological experiments in conjunction with confocal scanning laser microscopy and immune electron microscopy show that SmCyP is present in abundance in the adult worm as well as in the schistosomula. The function of CyP in the schistosome is presently unclear, but since its ligand, cyclosporin A, has antischistosomal activity, its function is expected to be a vital one.

Amino Acid Isomerases↗
PubMed1992

Characterization of human genomic yeast artificial chromosome inserts containing hexokinase 1 coding information on chromosome 10.

Hexokinase 1 (HK1) is one of four mammalian HK isoenzymes and maps to human chromosome 10. Two yeast artificial chromosomes (YACs) were identified in the Washington University human YAC library using polymerase chain reaction (PCR) primers designed with knowledge of the human HK1 cDNA sequence. YAC B129B12 is 120 kb in length and maps entirely to chromosome 10. YAC A159D5 is 400 kb in length and appears to have resulted from a recombination of chromosome 10 with non-chromosome 10 material. We report these YACs as potential resources for those interested in HK1 gene organization and mapping, as well as those desiring additional genomic information and markers on chromosome 10.

Animals↗
PubMed2003

Exploiting the yeast Saccharomyces cerevisiae for the study of the organization and evolution of complex genomes.

Yeast artificial chromosome (YAC) cloning systems have advanced the analysis of complex genomes considerably. They permit the cloning of larger fragments than do bacterial artificial chromosome systems, and the cloned material is more easily modified. We recently developed a novel YAC cloning system called transformation-associated recombination (TAR) cloning. Using in vivo recombination in yeast, TAR cloning selectively isolates, as circular YACs, desired chromosome segments or entire genes from complex genomes. The ability to do that without constructing a representative genomic library of random clones greatly facilitates analysis of gene function and its role in disease. In this review, we summarize how recombinational cloning techniques have advanced the study of complex genome organization, gene expression, and comparative genomics.

Chromosomes, Artificial, Yeast↗
PubMed2006

Sequence-independent helical wrapping of single-walled carbon nanotubes by long genomic DNA.

Because of their nanometer sizes and molecular recognition capabilities, biological systems have garnered much attention as vehicles for the directed assembly of nanoscale materials.(1-6) One of the greatest challenges of this research has been to successfully interface biological systems with electronic materials, such as semiconductors and metals. As a means to address some of these issues, Sarikaya, Belcher, and others have used a combinatorial technique called phage display(7-9) to discover new families of peptides that showed binding affinities to various substrates. More recently, Zheng and co-workers used combinatorial DNA libraries to isolate short DNA oligomers (30-90 bases) that could disperse single-walled carbon nanotubes (SWCNT) in water.(10) Through a systematic analysis, they found that short oligonucleotides having repeating sequences of gunanines and thymines (dGdT)(n) could wrap in a helical manner around a CNT with periodic pitch.(11) Although helix formation around SWCNTs having regular pitches is an effective method for dispersing and separating CNTs, the need for specific repeating sequences limits use to non-natural DNA that must be synthesized with optimal lengths of less than 150 bases. In contrast, we demonstrate here that long genomic single-stranded DNA (>>100 bases) of a completely random sequence of bases can be used to disperse CNTs efficiently through the single-stranded DNA's (ssDNA) ability to form tight helices around the CNTs with distinct periodic pitches. Although this process occurs irrespective of the DNA sequence, we show that this process is highly dependent on the removal of complementary strands. We also demonstrate that although the helix pitch-to-pitch distances remain constant down the length of a single CNT, the distances are variable from one DNA-CNT to another. Finally, we report initial work that shows that methods developed to align long dsDNA can be applied in a similar fashion to produce highly dense arrays of aligned ssDNA-CNT hybrids.

DNA, Single-Stranded↗
PubMed2005

A new ligand for immunoglobulin g subdomains by screening of a synthetic peptide library.

By screening a synthetic peptide library of general formula (NH(2)-Cys1-X2-X3-X4)(2)-Lys-Gly-OH, a disulfide-bridged cyclic peptide, where X2-X3-X4 is the tripeptide Phe-His-His, has been selected as a ligand for immunoglobulin G (IgG). The peptide, after a preliminary chromatographic characterization, has proved useful as a new affinity ligand for the purification of polyclonal as well as monoclonal antibodies from biological fluids, with recovery yields of up to 90% (90% purity). The ligand is able to bind antibody fragments containing both Fab and Fc from different antibody isotypes, a fact suggesting the presence of at least two different antibody-binding sites. While the recognition site on Fab is unknown, comparative binding studies with Fc, in association with the striking similarities of the peptide (named Fc-receptor mimetic, FcRM) with a region of the human FcgammaRIII receptor, strongly indicate that the peptide could recognize a short amino acid stretch of the lower hinge region, which has a key role in autoimmune disease triggering. The unique properties make the ligand attractive for both the purification of antibody fragments and as a lead for the generation of Fc-receptor antagonists.

Amino Acid Sequence↗
PubMed2000

Chromosomal organization and localization of the human histone deacetylase 5 gene (HDAC5).

Histone deacetylases (HDACs) are important participants in the remodeling of chromatin structure and in the regulation of eukaryotic proliferation and differentiation. We have isolated and characterized the human HDAC5 genomic sequence, which spans a region of 39,138 bp and which has one single chromosomal locus. Determination of the exon-intron splice junctions established that HDAC5 is encoded by 26 exons ranging in size from 22 bp (exon 1) to 285 bp (exon 12). Characterization of the 5' flanking genomic region revealed that the human HDAC5 promoter lacks both the canonical TATA and CCAAT boxes. The human HDAC5 mRNA encodes a 1122 aa protein with a predictive molecular mass of 121.9 kDa and an isoelectric point of 5.84. Fluorescence in situ hybridization analysis localized the human HDAC5 gene to chromosome 17q21, a region which is characterized by frequent gains and losses of chromosomal material in several types of cancer.

Amino Acid Sequence↗
PubMed1999

"Mutagenesis" by peptide aptamers identifies genetic network members and pathway connections.

We selected peptide aptamers from combinatorial libraries that disrupted cell-cycle arrest caused by mating pheromone in yeast. We used these aptamers as baits in two-hybrid hunts to identify genes involved in cell-cycle arrest. These experiments identified genes known to function in the pathway, as well as a protein kinase, the CBK1 product, whose function was not known. We used a modified two-hybrid system to identify specific interactions disrupted by these aptamers. These experiments demonstrate a means to perform "genetics" on the protein complement of a cell without altering its genetic material. Peptide aptamers can be identified that disrupt a process. These aptamers can then be used as affinity reagents to identify individual proteins and protein interactions needed for the process. Forward genetic analysis with peptide aptamer "mutagens" should be particularly useful in elucidating genetic networks in organisms and processes for which classical genetics is not feasible.

Amino Acid Sequence↗
PubMed2001

Haloperidol versus placebo for schizophrenia.

BACKGROUND: Haloperidol was developed in the late 1950s for use in the field of analgesia. Research subsequently demonstrated effects on hallucinations, delusions, aggressiveness, impulsiveness and states of excitement and led to the introduction of haloperidol as an antipsychotic. OBJECTIVES: To evaluate the clinical effects of haloperidol for the management of schizophrenia and other similar serious mental illnesses compared to placebo. SEARCH STRATEGY: Electronic searches of Biological Abstracts (1985-1998), CINAHL (1982-1998), The Cochrane Library (1998, Issue 4), The Cochrane Schizophrenia Group's Register (December 2000), EMBASE (1980-1998), MEDLINE (1966-1998), PsycLIT (1974-1998), and SCISEARCH (January 1974-December 1998) were undertaken. References of all identified studies were searched for further trial citations. Authors of trials and pharmaceutical companies were contacted for further information and archive material. SELECTION CRITERIA: All relevant randomised controlled trials comparing use of haloperidol (any dose) with placebo for those with schizophrenia or other similar serious, non-affective psychotic illnesses (however diagnosed). The main outcomes of interest were death, loss to follow up, clinical and social response, relapse and severity of adverse effects. DATA COLLECTION AND ANALYSIS: Reviewers evaluated data independently and analysed on an intention-to-treat basis, assuming that people who left the study early, or were lost to follow up, had no improvement. Where possible and appropriate, dichotomous data were analysed using relative risk (RR) and their 95% confidence intervals (CI) calculated. If appropriate, the number needed to treat (NNT) or number needed to harm (NNH) was estimated. For continuous data, weighted mean differences were calculated. Continuous data were excluded if loss to follow up was greater than 50%. All data were inspected for heterogeneity. MAIN RESULTS: Seventy-four trials were identified but only 20 included. More people allocated to haloperidol improved in the first six weeks of treatment than those given placebo (three trials, n=159, RR failing to produce a marked improvement 0.44 CI 0.3 to 0.6, NNT 3 CI 2 to 5). A further eight trials (n=313) also found a difference favouring haloperidol across the 6-24 week period (RR no marked global improvement 0.68 CI 0.6 to 0.8 NNT 3 CI 2.5 to 5) but this may be an overestimate of effect as small negative studies were not identified. About half of those entering studies failed to complete the short trials, although, at 0-6 weeks, 10 studies found a difference that favoured haloperidol (n=686, RR 0.82 CI 0.7 to 0.95, NNT 8 CI 5 to 17). Limited adverse effect data do, nevertheless, support the clinical impression that haloperidol is a potent cause of movement disorders, at least in the short term. Haloperidol promotes acute dystonia (three trials, n=135, RR 4.7 CI 1.7 to 44, NNH 5 CI 3 to 9 - not assuming those who left early from placebo suffered dystonis), akathisia (three trials, n=129, RR 6.5 CI 1.5 to 28, NNH 6 CI 4 to 14) and parkinsonism (four trials, n=165, RR 8.9 CI 2.6 to 31, NNH 3 CI 2 to 5). REVIEWER'S CONCLUSIONS: Haloperidol is a potent antipsychotic drug but with a high propensity to cause adverse effects. Given no choice of drug, use of haloperidol to counter the damaging and potentially dangerous consequences of untreated schizophrenia is justified. If a choice of drug is available, however, people with schizophrenia and clinicians may wish to start another antipsychotic with less likelihood of causing parkinsonism, akathisia and acute dystonias. For countries where haloperidol is not widely used, it should not be a control drug of choice for randomised trials of new antipsychotics.

Antipsychotic Agents↗
PubMed1996

Antisense RNA Amplification: A Linear Amplification Method for Analyzing the mRNA Population from Single Living Cells

Cell-specific gene expression is a fundamental area of investigation of various disciplines, but is often complicated by cell heterogeneity, paucity of material, or limits of detection for low-abundance mRNAs. Antisense RNA (aRNA) amplification presents a linear amplification method for analyzing the mRNA population from single living cells. Patch clamp electrodes containing reverse transcriptase, dNTPS, and a poly(T) primer modified 5' with a T7 RNA polymerase promoter sequence are used to isolate the cytoplasmic contents of individual living cells. The cDNAs transcribed therefore contain the T7 promoter sequence. Following subsequent processing for second-strand cDNA synthesis, T7 polymerase is used for amplification, which results in a 2000-fold amplification of antisense RNA. A second round of amplification results in a 10(6)-fold amplification of the initial material. The aRNA amplification method may be used in conjunction with other techniques. Electrophysiology may be conducted first to examine functional properties. Reverse Northerns may be performed, using the aRNA as a probe to identify specific cDNAs loaded onto a slot blot. PCR may be conducted to determine splice variants. Differential display and library construction may be employed to identify unknown or novel genes. Thus, the aRNA amplification may prove a valuable method for providing information on cell-specific gene expression in a variety of studies.

Journal Article↗
PubMed1996

The identification of a novel cDNA preferentially expressed in the olfactory-limbic system of the adult rat.

To analyze cell-specific brain gene expression, we have developed a PCR-based subtractive hybridization cloning method utilizing trace starting material, allowing isolation of novel genes expressed under specific conditions. Our previous studies indicated that local substantia nigra (SN) type 1 astrocytes elaborate an array of trophic molecules which support the survival of SN dopaminergic neurons. Therefore, the current study focused on astrocyte gene expression utilizing a type 1 astrocyte-enriched cDNA library. We report initial characterization of a novel cDNA, designated AT1-46, that is preferentially expressed in the olfactory-limbic system of the adult rat brain. Although AT1-46 is expressed widely in the periphery, it is regulated both developmentally and in a cell-specific fashion in the brain. Structurally, AT1-46 is predicted to encode a highly alpha-helical molecule with several domains of potential coiled coil formation, and exhibits a 28% amino acid sequence identity with the intermediate filament-associated protein, trichohyalin.

Amino Acid Sequence↗
PubMed2004

RNA fingerprinting--a new method to screen for differences in plant litter degrading microbial communities.

Microbial activities are essential for the nutrient turnover processes in soil and play an important role in the degradation of complex organic material, for example, plant leaf litter. However, very little is known about the microorganisms and their genes involved during the course of leaf litter decomposition. In the present study, we describe the non-radioactive application of RNA arbitrarily primed-PCR (RAP-PCR) protocol in combination with the classic litter bag technique to investigate the metabolic profiles of microbial community involved in leaf litter degradation after 2 and 8 weeks of degradation in four different soil sites, without using selective primer systems for PCR. Due to the significantly reduced target sites for PCR primers, compared to the published papers about RAP fingerprinting of more complex microbial communities based on DNA analysis (only transcripts from microbes on the litter material were analysed), the patterns of parallel samples were highly reproducible (>95%). Shifts in microbial community structure and function were observed during the course of degradation. Each litter sample had its unique metabolic profile and both soil effects and litter quality effects were evident. RAP-PCR products were also cloned to generate libraries. Clone libraries were screened by restriction fragment length polymorphism (RFLP) and representative samples sequenced to identify the inserts. Both mRNA and rRNA transcripts were obtained confirming the presence of mRNA in total RNA preparations. Hence, the described protocol is a good screening method to find similarities or differences in the structure and function of microbial communities involved in litter degradation, which may be the basis for more detailed studies by cloning and sequencing approaches.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Expression of Jagged1 gene in macrophages and its regulation by hematopoietic growth factors.

OBJECTIVE: Serrate/Jagged and Delta are cell surface ligands for Notch receptors that may influence hematopoietic cell fate decisions and are known to be expressed in bone marrow stromal cells. In a series of screenings of cDNAs constructed by a cDNA library subtraction technique, we identified Jagged1, one of the Notch ligands, as a gene up-regulated by macrophage colony-stimulating factor (M-CSF) in bone marrow macrophages. Therefore, we compared stromal cells and macrophages for expression of Notch ligands including Jagged1 and analyzed the regulation of their expression by cytokines. MATERIALS AND METHODS: Murine bone marrow macrophages were prepared by culturing femoral bone marrow cells with M-CSF. Primary bone marrow fibroblastic stromal cells were prepared by a culture system that we recently developed. The expression of Notch ligands was analyzed by either Northern blot analysis or reverse transcriptase polymerase chain reaction. RESULTS: The bone marrow macrophages expressed Jagged1 but not Jagged2 and Delta1 at a level that was detectable by Northern blot analysis. Expression of the Jagged1 gene was markedly up-regulated by growth factors for the cells, i.e., M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. Expression of Jagged2 and Delta1 seldom was affected by the stimuli. The primary bone marrow fibroblastic stromal cells, and murine stromal cell lines, such as PA6 and ST2, also expressed Jagged1 transcript, at levels comparable to the steady-state level in macrophages. However, expression of the Jagged1 gene was little affected when these cells were stimulated with fibroblastic growth factor and platelet-derived growth factor. CONCLUSIONS: We demonstrated that bone marrow macrophages as well as stromal cells constitutively produced Jagged1 and that the expression was markedly up-regulated by hematopoietic growth factors, M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. The results highlight the involvement of macrophages and these growth factors in hematopoietic cell fate decisions via the production of Jagged1.

Animals↗
PubMed2003

Expression analysis of subtractively enriched libraries (EASEL): a widely applicable approach to the identification of differentially expressed genes.

A variety of methods for high throughput analysis of differential gene expression has been developed over the past years. We have implemented the EASEL technique that adds flexibility, efficiency and wide-applicability to these methods. The EASEL procedure is unique as it integrates several well established techniques and thereby offers a combination of subtractive hybridization of 3' cDNA ends with macroarrays analysis and Serial Analysis of Gene Expression (SAGE). In addition, once a set of interesting, differentially expressed genes is identified, the material required for follow up studies to test the hypothesis that the gene is truly involved in the process of interest is readily available. In this report, we first present a step-by-step validation of the procedure, since several of the incorporated steps had to be tailored to meet specific requirements and implied drastic modifications of the original methods. Secondly, we applied EASEL to the identification of up-regulated gene products in the outflow tract region of the embryonic rat heart. Here we provide evidence that at least two among the differentially expressed genes detected, follistatin-like protein gene and membrane type 1-metallo proteinase gene, are selectively up-regulated in the outflow tract, suggesting their involvement in the development of this region during embryogenesis.

Animals↗
PubMed2000

Creating addressable aqueous microcompartments above solid supported phospholipid bilayers using lithographically patterned poly(dimethylsiloxane) molds

Herein we report the use of microcontact displacement (microCD) to generate addressable arrays of aqueous solutions above fluid lipid bilayers by bringing a patterned PDMS mold into conformal contact with a phospholipid membrane on a solid supported substrate. Epifluorescence microscopy established that the bilayer material was displaced in regions where contact between the mold and substrate was made. Photobleaching experiments confirmed that the bilayer sectors remained individually fluid but completely separated. The microcompartments created by microCD could be individually injected with aqueous solutions that remained sealed from their neighbors. This procedure was then exploited for screening a small library of aqueous-phase molecules for their ability to inhibit binding between surface-bound ligands and soluble protein receptors.

Journal Article↗
PubMed1990

Use of pulsed field gel electrophoresis to differentiate Coxiella burnetii strains.

Pulsed field gradient gel electrophoresis (PFGE) provides a powerful technique for the analysis of bacterial genomic DNA by allowing the resolution of DNA fragments as large as 9000 kilobase pairs (kbp). When isolates of Coxiella burnetii were examined using this method, the restriction enzymes Not I and Sfi I gave the fewest and most easily resolved fragments. Sfi I cuts the genome of the Priscilla isolate of C. burnetii into 15 DNA fragments ranging in size from 320 to 18 kbp, and Not I cuts the DNA of this isolate into 20 fragments from 293 to 10 kbp in size. Analysis of the undigested DNA and summing of the Sfi I restriction fragments both indicate that the C. burnetii DNA contains approximately 1600 kbp, or is about one-third the size of the DNA in Escherichia coli. Comparisons of isolates revealed that the numbers and patterns of DNA fragments observed correlate with proposed strain designations. Because PFGE allows the reproducible separation of restriction endonuclease-digested C. burnetii DNA fragments into precise bands, it greatly facilitates the selection of large DNA fragments for cloning. Bands harvested from the gel can be cloned. Clone banks are invaluable for identifying the location of specific genes and landmarks and providing material for future experiments, including DNA sequencing. Yeast artificial chromosome (YAC) cloning vectors can accept fragments as large as 500 kbp. The fragmentation patterns of C. burnetii that we have obtained with infrequent-cutting enzymes are small enough to be cloned into YAC vectors. Using a PFGE selection method means that only small libraries would have to be created and screened. Thus, the results of these experiments also demonstrate the applicability of PFGE for deriving a physical map of C. burnetii chromosomal DNA. Development of such a macrorestriction map will facilitate genetic and molecular studies with C. burnetii.

Coxiella↗