A long-range regulatory element of Hoxc8 identified by using the pClasper vector.

Hox genes are located in highly conserved clusters. The significance of this organization is unclear, but one possibility is that regulatory regions for individual genes are dispersed throughout the cluster and shared with other Hox genes. This hypothesis is supported by studies on several Hox genes in which even large genomic regions immediately surrounding the gene fail to direct the complete expression pattern in transgenic mice. In particular, previous studies have identified proximal regulatory regions that are primarily responsible for early phases of mouse Hoxc8 expression. To locate additional regulatory regions governing expression during the later periods of development, a yeast homologous recombination-based strategy utilizing the pClasper vector was employed. Using homologous recombination into pClasper, we cloned a 27-kb region around the Hoxc8 gene from a yeast artificial chromosome. A reporter gene was introduced into the coding region of the isolated gene by homologous recombination in yeast. This large fragment recapitulates critical aspects of Hoxc8 expression in transgenic mice. We show that the regulatory elements that maintain the anterior boundaries of expression in the neural tube and paraxial mesoderm are located between 11 and 19 kb downstream of the gene.

A family of transmembrane proteins with homology to the MET-hepatocyte growth factor receptor.

In hunting for unknown genes on the human X chromosome, we identified a cDNA in Xq28 encoding a transmembrane protein (SEX) of 1871 amino acids. SEX shares significant homology with the extracellular domain of the receptors encoded by the oncogenes MET, RON, and SEA [hepatocyte growth factor (HGF) receptor family]. Further screenings of cDNA libraries identified three additional sequences closely related to SEX: these were named SEP, OCT, and NOV and were located on human chromosomes 3p, 1, and 3q, respectively. The proteins encoded by these genes contain large cytoplasmic domains characterized by a distinctive highly conserved sequence (SEX domain). Northern blot analysis revealed different expression of the SEX family of genes in fetal tissues, with SEX, OCT, and NOV predominantly expressed in brain, and SEP expressed at highest levels in kidney. In situ hybridization analysis revealed that SEX has a distinctive pattern of expression in the developing nervous system of the mouse, where it is found in postmitotic neurons from the first stages of neuronal differentiation (9.5 day postcoitus). The SEX protein (220 kDa) is glycosylated and exposed at the cell surface. Unlike the receptors of the HGF family, p220SEX, a MET-SEX chimera or a constitutively dimerized TPR-SEX does not show tyrosine kinase activity. These data define a gene family (SEX family) involved in the development of neural and epithelial tissues, which encodes putative receptors with unexpected enzymatic or binding properties.

Blockade of T- and B-lymphocyte development by antibody to the gamma c subunit of the receptors for interleukins 2, 4, and 7.

Cytokines are important regulators of hematopoesis. Mutations in gamma c, which is a subunit shared by the receptors for interleukin (IL) 2, IL-4, and IL-7, have been causally associated with human X chromosome-linked severe combined immunodeficiency disease. This finding indicates a mandatory role for cytokine receptor signaling at one or more stages of lymphocyte development. To evaluate the cellular level at which gamma c is critical for lymphopoiesis, the effect of monoclonal antibodies to gamma c on the capacity of syngeneic bone marrow cells to reconstitute the hematopoietic compartment of lethally irradiated recipient mice was examined. We show that monoclonal antibody to gamma c blocked lymphocyte development at or before the appearance of pro-B cells and prior to or at the seeding of the thymus by precursor cells while erythromyeloid cell development was normal. These results suggest that one level of lymphocyte development that requires gamma c is a point in hematopoietic cell differentiation near the divergence of lymphopoiesis and erythromyelopoesis.

Sequencing a new target genome: The Boophilus microplus (Acari : Ixodidae) genome project

The southern cattle tick, Boophilus microplus (Canestrini), causes annual economic losses in the hundreds of millions of dollars to cattle producers throughout the world, and ranks as the most economically important tick from a global perspective. Control failures attributable to the development of pesticide resistance have become commonplace, and novel control technologies are needed. The availability of the genome sequence will facilitate the development of these new technologies, and we are proposing sequencing to a 4-6X draft coverage. Many existing biological resources are available to facilitate a genome sequencing project, including several inbred laboratory tick strains, a database of approximate to 45,000 expressed sequence tags compiled into a B. microplus Gene Index, a bacterial artificial chromosome (BAC) library, an established B. microplus cell line, and genomic DNA suitable for library synthesis. Collaborative projects are underway to map BACs and cDNAs to specific chromosomes and to sequence selected BAC clones. When completed, the genome sequences from the cow, B. microphis, and the B. microphis-borne pathogens Babesia bovis and Anaplasma marginale will enhance studies of host-vector-pathogen systems. Genes involved in the regeneration of amputated tick limbs and transitions through developmental stages are largely unknown. Studies of these and other interesting biological questions will be advanced by tick genome sequence data. Comparative genomics offers the prospect of new insight into many, perhaps all, aspects of the biology of ticks and the pathogens they transmit to farm animals and people. The B. microplus genome sequence will fill a major gap in comparative genomics: a sequence from the Metastriata lineage of ticks. The purpose of the article is to synergize interest in and provide rationales for sequencing the genome of B. microplus and for publicizing currently available genomic resources for this tick.

Microarray-based comparative genomic hybridisation of breast cancer patients receiving neoadjuvant chemotherapy

We analysed the molecular genetic profiles of breast cancer samples before and after neoadjuvant chemotherapy with combination doxorubicin and cyclophosphamide (AC). DNA was obtained from microdissected frozen breast core biopsies from 44 patients before chemotherapy. Additional samples were obtained before the second course of chemotherapy (D21) and after the completion of the treatment (surgical specimens) in 17 and 21 patients, respectively. Microarray-based comparative genome hybridisation was performed using a platform containing approx5800 bacterial artificial chromosome clones (genome-wide resolution: 0.9 Mb). Analysis of the 44 pretreatment biopsies revealed that losses of 4p, 4q, 5q, 12q13.11–12q13.12, 17p11.2 and 17q11.2; and gains of 1p, 2p, 7q, 9p, 11q, 19p and 19q were significantly associated with oestrogen receptor negativity. 16q21–q22.1 losses were associated with lobular and 8q24 gains with ductal types. Losses of 5q33.3–q4 and 18p11.31 and gains of 6p25.1–p25.2 and Xp11.4 were associated with HER2 amplification. No correlations between DNA copy number changes and clinical response to AC were found. Microarray-based comparative genome hybridisation analysis of matched pretreatment and D21 biopsies failed to identify statistically significant differences, whereas a comparison between matched pretreatment and surgical samples revealed a statistically significant acquired copy number gain on 11p15.2–11p15.5. The modest chemotherapy-driven genomic changes, despite profound loss of cell numbers, suggest that there is little therapeutic selection of resistant non-modal cell lineages.

Cloning of the ω-secalin gene family in a wheat 1BL/1RS translocation line using BAC clone sequencing

Background: Wheat 1BL/1RS translocation lines are planted around the world for their disease resistance and high yield. Most of them are poor in bread making, which is partially caused by ω-secalins that are encoded by the ω-secalin gene family, which is located on the short arm of rye chromosome 1R (1RS). However, information on the structure and evolution of the ω-secalin gene family is still limited. Results: We first generated a physicalmap of the ω-secalin gene family covering 195 kb of the Sec-1 locus based on sequencing three bacterial artificial chromosome (BAC) clones of the 1BL/1RS translocation wheat cultivar Shimai 15. A BAC contig was constructed spanning 168 kb of the Sec-1 locus on 1RS. Twelve ω-secalin genes were arranged in a head-to-tail fashion, separated by 8.2–21.6 kb spacers on the contig, whereas six other ω-secalin genes were arranged head-to-tail, separated by 8.2–8.4 kb of spacers on clone BAC125. The 18 ω-secalin genes can be classified into six types among which eight ω-secalin genes were expressed during seed development. The ω-secalin genes with the 1074-bp open reading frame (ORF) represented the main population. Except for two pseudogenes, the N-terminal of the ω-secalin gene was conserved, whereas variations in the C-terminal led to a change in ORF length. The spacers can be sorted into two classes. Class-1 spacers contained conserved and non-conservative sequences. Conclusion: The ω-secalin gene family consisted of at least 18 members in the 1BL/1RS translocation line cv. Shimai 15. Eight ω-secalin genes were expressed during seed development. Eighteen members may originate from a progenitor with a 1,074-bp ORF. The spacers differed in length and sequence conservation.

Identificação de genes MUTM em BACs da cana-de-açucar e caracterização preliminar destes genes e seus promotores

Sugarcane has an importance in Brazil due to sugar and biofuel production. Considering this aspect, there is basic research being done in order to understand its physiology to improve production. The aim of this research is the Base Excision Repair pathway, in special the enzyme MUTM DNA-glycosylase (formamidopyrimidine) which recognizes oxidized guanine in DNA. The sugarcane scMUTM genes were analyzed using four BACs (Bacterial Artificial Chromosome) from a sugarcane genomic library from R570 cultivar. The resulted showed the presence in the region that had homology to scMUTM the presence of transposable elements. Comparing the similarity, it was observed a highest similarity to Sorghum bicolor sequence, both nucleotide and peptide sequences. Furthermore, promoter regions from MUTM genes in some grass showed different cis-regulatory elements, among which, most were related to oxidative stress, suggesting a gene regulation by oxidative stress

Analysis of contributions of herpes simplex virus type 1 UL43 protein to induction of cell-cell fusion

Purpose: To investigate whether UL43 protein, which is highly conserved in alpha- and gamma herpes viruses, and a non-glycosylated transmembrane protein, is involved in virus entry and virus-induced cell fusion. Methods: Mutagenesis was accomplished by a markerless two-step Red recombination mutagenesis system implemented on the Herpes simplex virus 1 (HSV-1) bacterial artificial chromosome (BAC). Growth properties of HSV-1 UL43 mutants were analyzed using plaque morphology and one-step growth kinetics. SDS-PAGE and Western blot was employed to assay the synthesis of the viral glycoproteins. Virus-penetration was assayed to determine if UL43 protein is required for efficient virus entry. Results: Lack of UL43 expression resulted in significantly reduced plaque sizes of syncytial mutant viruses and inhibited cell fusion induced by gBΔ28 or gKsyn20 (p < 0.05). Deletion of UL43 did not affect overall expression levels of viral glycoproteins gB, gC, gD, and gH on HSV-1(F) BAC infected cell surfaces. Moreover, mutant viruses lacking UL43 gene exhibited slower kinetics of entry into Vero cells than the parental HSV-1(F) BAC. Conclusion: Thus, these results suggest an important role for UL43 protein in mediating virus-induced membrane fusion and efficient entry of virion into target cells.

Generation and screening of natural product-like compounds for antibiotic discovery

Avec l’apparition de plus en plus de souches de bactérie résistante aux antibiotiques, le développement de nouveaux antibiotiques est devenu une important problématique pour les agences de santé. C’est pour cela que la création de nouvelles plateformes pour accélérer la découverte de médicaments est devenu un besoin urgent. Dans les dernières décennies, la recherche était principalement orientée sur la modification de molécules préexistantes, la méta-analyse d’organismes produisant des molécules activent et l’analyse de librairies moléculaires pour trouver des molécules synthétiques activent, ce qui s’est avéré relativement inefficace. Notre but était donc de développer de nouvelles molécules avec des effets thérapeutiques de façon plus efficace à une fraction du prix et du temps comparé à ce qui se fait actuellement. Comme structure de base, nous avons utilisé des métabolites secondaires qui pouvaient altérer le fonctionnement des protéines ou l’interaction entre deux protéines. Pour générer ces molécules, j’ai concentré mes efforts sur les terpènes, une classe de métabolites secondaires qui possède un large éventail d’activités biologiques incluant des activités antibactériennes. Nous avons développé un système de chromosome artificiel de levure (YAC) qui permet à la fois l’assemblage directionnel et combinatoire de gènes qui permet la création de voies de biosynthèse artificielles. Comme preuve de concept, j’ai développé des YACs qui contiennent les gènes pour l’expression des enzymes impliquées dans la biosynthèse de la -carotène et de l’albaflavenone et produit ces molécules avec un haut rendement. Finalement, Des YACs produits à partir de librairies de gènes ont permis de créer une grande diversité de molécules.

Generation and screening of natural product-like compounds for antibiotic discovery

Avec l’apparition de plus en plus de souches de bactérie résistante aux antibiotiques, le développement de nouveaux antibiotiques est devenu une important problématique pour les agences de santé. C’est pour cela que la création de nouvelles plateformes pour accélérer la découverte de médicaments est devenu un besoin urgent. Dans les dernières décennies, la recherche était principalement orientée sur la modification de molécules préexistantes, la méta-analyse d’organismes produisant des molécules activent et l’analyse de librairies moléculaires pour trouver des molécules synthétiques activent, ce qui s’est avéré relativement inefficace. Notre but était donc de développer de nouvelles molécules avec des effets thérapeutiques de façon plus efficace à une fraction du prix et du temps comparé à ce qui se fait actuellement. Comme structure de base, nous avons utilisé des métabolites secondaires qui pouvaient altérer le fonctionnement des protéines ou l’interaction entre deux protéines. Pour générer ces molécules, j’ai concentré mes efforts sur les terpènes, une classe de métabolites secondaires qui possède un large éventail d’activités biologiques incluant des activités antibactériennes. Nous avons développé un système de chromosome artificiel de levure (YAC) qui permet à la fois l’assemblage directionnel et combinatoire de gènes qui permet la création de voies de biosynthèse artificielles. Comme preuve de concept, j’ai développé des YACs qui contiennent les gènes pour l’expression des enzymes impliquées dans la biosynthèse de la -carotène et de l’albaflavenone et produit ces molécules avec un haut rendement. Finalement, Des YACs produits à partir de librairies de gènes ont permis de créer une grande diversité de molécules.

A novel myeloid leukemia suppressor locus at human chromosome 5q13.3

Molecular mechanisms that underlie preleukemic myelodysplasia (MDS) and acute myelogenous leukemia (AML) are poorly understood. In MDS or AML with a refractory clinical course, more than 30% of patients have acquired interstitial or complete deletions of chromosome 5. The 5q13.3 chromosomal segment is commonly lost as the result of 5q deletion. Reciprocal and unbalanced translocations of 5q13.3 can also occur as sole anomalies associated with refractory AML or MDS. This study addresses the hypothesis that a critical gene at 5q13.3 functions either as a classical tumor suppressor or as a chromosomal translocation partner and contributes to leukemogenesis. ^ Previous studies from our laboratory delineated a critical region of loss to a 2.5–3.0Mb interval at 5q13.3 between microsatellite markers D5S672 and GATA-P18104. The critical region of loss was later resolved to an interval of approximately 2Mb between the markers D5S672 and D5S2029. I, then generated a long range physical map of yeast artificial chromosomes (YACs) and developed novel sequence tagged sites (STS). To enhance the resolution of this map, bacterial artificial chromosomes (BACs) were used to construct a triply linked contig across a 1 Mb interval. These BACs were used as probes for fluorescent in situ hybridization (FISH) on an AML cell line to define the 5q13.3 critical region. A 200kb BAC, 484a9, spans the translocation breakpoint in this cell line. A novel gene, SSDP2 (single stranded DNA binding protein), is disrupted at the breakpoint because its first four exons are encoded within 140kb of BAC 484a9. This finding suggests that SSDP2 is the critical gene at 5q13.3. ^ In addition, I made an observation that deletions of chromosome 5q13 co-segregate with loss of the chromosome 17p. In some cases the deletions result from unbalanced translocations between 5q13 and 17p13. It was confirmed that the TP53 gene is deleted in patients with 17p loss, and the remaining allele harbors somatic mutation. Thus, the genetic basis for the aggressive clinical course in AML and MDS may be caused by functional cooperation between deletion or disruption of the 5q13.3 critical gene and inactivation of TP53. ^

Direct isolation of human transcribed sequences from yeast artificial chromosomes through the application of RNA fingerprinting

The identification of cDNA clones from genomic regions known to contain human genes is usually the rate-limiting factor in positional cloning strategies. We demonstrate here that human genes present on yeast artificial chromosomes (YACs) are transcribed in yeast host cells. We have used the arbitrarily primed RNA (RAP) fingerprinting method to identify human-specific, transcribed sequences from YACs located in the 13q12 chromosome region. By comparing the RAP fingerprints generated using defined, arbitrary primers from various fragmented YACs, megaYACs, and host yeast, we were able to identify and map 20 products transcribed from the human YAC inserts. This method, therefore, permits the simultaneous isolation and mapping of novel expressed sequences directly from whole YACs.

Comparative analysis of the gene-dense ACHE/TFR2 region on human chromosome 7q22 with the orthologous region on mouse chromosome 5

Chromosome 7q22 has been the focus of many cytogenetic and molecular studies aimed at delineating regions commonly deleted in myeloid leukemias and myelodysplastic syndromes. We have compared a gene-dense, GC-rich sub-region of 7q22 with the orthologous region on mouse chromosome 5. A physical map of 640 kb of genomic DNA from mouse chromosome 5 was derived from a series of overlapping bacterial artificial chromosomes. A 296 kb segment from the physical map, spanning Ache to Tfr2, was compared with 267 kb of human sequence. We identified a conserved linkage of 12 genes including an open reading frame flanked by Ache and Asr2, a novel cation-chloride cotransporter interacting protein Cip1, Ephb4, Zan and Perq1. While some of these genes have been previously described, in each case we present new data derived from our comparative sequence analysis. Adjacent unfinished sequence data from the mouse contains an orthologous block of 10 additional genes including three novel cDNA sequences that we subsequently mapped to human 7q22. Methods for displaying comparative genomic information, including unfinished sequence data, are becoming increasingly important. We supplement our printed comparative analysis with a new, Web-based program called Laj (local alignments with java). Laj provides interactive access to archived pairwise sequence alignments via the WWW. It displays synchronized views of a dot-plot, a percent identity plot, a nucleotide-level local alignment and a variety of relevant annotations. Our mouse–human comparison can be viewed at http://web.uvic.ca/~bioweb/laj.html. Laj is available at http://bio.cse.psu.edu/, along with online documentation and additional examples of annotated genomic regions.

A compositional map of human chromosome band Xq28.

The molar fractions of guanine plus cytosine (GC) in DNA were determined for 36 yeast artificial chromosomes (YACs) which almost completely cover human chromosome band Xq28, a terminal reverse band, corresponding to about 8 Mb of DNA. This allowed the construction of the most complete compositional map to date of a chromosomal band; three regions were observed: (i) a proximal 3.5-Mb region formed by GC-poor L and GC-rich H1 isochores; (ii) a middle 2,2-Mb region essentially formed by a GC-rich H2 isochore and a very GC-rich H3 isochore separated by a GC-poor L isochore, YACs from this region being characterized by a striking compositional heterogeneity and instability; and (iii) a distal 1.3-Mb region exclusively formed by GC-poor L isochores. Gene and CpG island concentrations increased with the GC levels of the isochores, as expected. Xq28 exemplifies a subset of reverse bands which are different from the two other subsets, namely from telomeric bands, which are characterized by specific cytogenetic properties and by the predominance of H2 and H3 isochores, and from the majority of reverse bands, which do not contain H2 and H3 isochores.

Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination.

DNA molecules undergoing transformation into yeast are highly recombinogenic, even when diverged. We reasoned that transformation-associated recombination (TAR) could be employed to clone large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation and reducing the amount of DNA handling. Gently isolated human DNA was transformed directly into yeast spheroplasts along with two genetically marked (M1 and M2) linearized vectors that contained a human Alu sequence at one end and a telomere sequence at the other end (Alu-CEN-M1-TEL and Alu-M2-TEL). Nearly all the M1-selected transformants had yeast artificial chromosomes (YACs) containing human DNA inserts that varied in size from 70 kb to > 600 kb. Approximately half of these had also acquired the unselected M2 marker. The mitotic segregational stability of YACs generated from one (M1) or two (M1 and M2) vector(s) was comparable, suggesting de novo generation of telomeric ends. Since no YACs were isolated when rodent DNAs or a vector lacking an Alu sequence was used, the YACs were most likely the consequence of TAR between the repeat elements on the vector(s) and the human DNA. Using the BLUR13 Alu-containing vector, we demonstrated that human DNA could be efficiently cloned from mouse cells that contained a single human chromosome 16. The distribution of cloned DNAs on chromosome 16 was determined by fluorescence in situ hybridization. We propose that TAR cloning can provide an efficient means for generating YACs from specific chromosomes and subchromosome fragments and that TAR cloning may be useful for isolating families of genes and specific genes from total genome DNA.