A novel multigene family encodes diversified variable regions

Antigen recognition in the adaptive immune response by Ig and T-cell antigen receptors (TCRs) is effected through patterned differences in the peptide sequence in the V regions. V-region specificity forms through genetically programmed rearrangement of individual, diversified segmental elements in single somatic cells. Other Ig superfamily members, including natural killer receptors that mediate cell-surface recognition, do not undergo segmental reorganization, and contain type-2 C (C2) domains, which are structurally distinct from the C1 domains found in Ig and TCR. Immunoreceptor tyrosine-based inhibitory motifs that transduce negative regulatory signals through the cell membrane are found in certain natural killer and other cell surface inhibitory receptors, but not in Ig and TCR. In this study, we employ a genomic approach by using the pufferfish (Spheroides nephelus) to characterize a nonrearranging novel immune-type receptor gene family. Twenty-six different nonrearranging genes, which each encode highly diversified V as well as a V-like C2 extracellular domain, a transmembrane region, and in most instances, an immunoreceptor tyrosine-based inhibitory motif-containing cytoplasmic tail, are identified in an ≈113 kb P1 artificial chromosome insert. The presence in novel immune-type receptor genes of V regions that are related closely to those found in Ig and TCR as well as regulatory motifs that are characteristic of inhibitory receptors implies a heretofore unrecognized link between known receptors that mediate adaptive and innate immune functions.

Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning

The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was ≥60 bp were utilized, ∼2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is ∼60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.

The rice genome project in Japan

Since 1991, the Rice Genome Research Program in Japan has carried out rice genomics, such as large-scale cDNA analysis, construction of a fine-scale restriction fragment length polymorphism map, and physical mapping of the rice genome with yeast artificial chromosome clones. These studies have made a great impact on research into grass genomes and made rice a model plant for other cereal crop research. Starting in 1998, the Rice Genome Research Program will step into a new stage of genomics—that of genome sequencing. This project eventually should reveal all of the genomic sequence information in the rice plant and be an indispensable aid in understanding the genomics of other grass species.

Hd6, a rice quantitative trait locus involved in photoperiod sensitivity, encodes the α subunit of protein kinase CK2

Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the japonica variety Nipponbare and the indica variety Kasalath. To isolate a gene at Hd6, we used a large segregating population for the high-resolution and fine-scale mapping of Hd6 and constructed genomic clone contigs around the Hd6 region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited Hd6 to a 26.4-kb genomic region. We identified a gene encoding the α subunit of protein kinase CK2 (CK2α) in this region. The Nipponbare allele of CK2α contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of CK2α increases days-to-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype.

Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF.

Globin genes are subject to tissue-specific and developmental stage-specific regulation. A switch from human fetal (gamma)-to adult (beta)-globin expression occurs within erythroid precursor cells of the adult lineage. Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Krüppel-like factor (EKLF), is required for expression of the beta-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter. To examine the role of EKLF in the developmental regulation of the human gamma-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human beta-globin yeast artificial chromosome transgene. We find that in the absence of EKLF, while human beta-globin expression is dramatically reduced, gamma-globin transcripts are elevated approximately 5-fold. Impaired silencing of gamma-globin expression identifies EKLF as the first transcription factor participating quantitatively in the gamma-globin to beta-globin switch. Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the beta-globin gene promoter and the locus control region that excludes the gamma-globin gene.

Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.

Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

Genomic cloning of methylthioadenosine phosphorylase: a purine metabolic enzyme deficient in multiple different cancers.

5'-Deoxy-5'-methylthioadenosine phosphorylase (methylthioadeno-sine: ortho-phosphate methylthioribosyltransferase, EC 24.2.28; MTAP) plays a role in purine and polyamine metabolism and in the regulation of transmethylation reactions. MTAP is abundant in normal cells but is deficient in many cancers. Recently, the genes for the cyclin-dependent kinase inhibitors p16 and p15 have been localized to the short arm of human chromosome 9 at band p21, where MTAP and interferon alpha genes (IFNA) also map. Homozygous deletions of p16 and p15 are frequent malignant cell lines. However, the order of the MTAP, p16, p15, and IFNA genes on chromosome 9p is uncertain, and the molecular basis for MTAP deficiency in cancer is unknown. We have cloned the MTAP gene, and have constructed a topologic map of the 9p21 region using yeast artificial chromosome clones, pulse-field gel electrophoresis, and sequence-tagged-site PCR. The MTAP gene consists of eight exons and seven introns. Of 23 malignant cell lines deficient in MTAP protein, all but one had complete or partial deletions. Partial or total deletions of the MTAP gene were found in primary T-cell acute lymphoblastic leukemias (T-ALL). A deletion breakpoint of partial deletions found in cell lines and primary T-ALL was in intron 4. Starting from the centromeric end, the gene order on chromosome 9p2l is p15, p16, MTAP, IFNA, and interferon beta gene (IFNB). These results indicate that MTAP deficiency in cancer is primarily due to codeletion of the MTAP and p16 genes.

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.

Mapping the mouse genome: current status and future prospects.

The mouse is the best model system for the study of mammalian genetics and physiology. Because of the feasibility and importance of studying genetic crosses, the mouse genetic map has received tremendous attention in recent years. It currently contains over 14,000 genetically mapped markers, including 700 mutant loci, 3500 genes, and 6500 simple sequence length polymorphisms (SSLPs). The mutant loci and genes allow insights and correlations concerning physiology and development. The SSLPs provide highly polymorphic anchor points that allow inheritance to be traced in any cross and provide a scaffold for assembling physical maps. Adequate physical mapping resources--notably large-insert yeast artificial chromosome (YAC) libraries--are available to support positional cloning projects based on the genetic map, but a comprehensive physical map is still a few years away. Large-scale sequencing efforts have not yet begun in mouse, but comparative sequence analysis between mouse and human is likely to provide tremendous information about gene structure and regulation.

Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore

Kinetochores assemble on distinct 'centrochromatin' containing the histone H3 variant CENP-A and interspersed nucleosomes dimethylated on H3K4 (H3K4me2). Little is known about how the chromatin environment at active centromeres governs centromeric structure and function. Here, we report that centrochromatin resembles K4-K36 domains found in the body of some actively transcribed housekeeping genes. By tethering the lysine-specific demethylase 1 (LSD1), we specifically depleted H3K4me2, a modification thought to have a role in transcriptional memory, from the kinetochore of a synthetic human artificial chromosome (HAC). H3K4me2 depletion caused kinetochores to suffer a rapid loss of transcription of the underlying α-satellite DNA and to no longer efficiently recruit HJURP, the CENP-A chaperone. Kinetochores depleted of H3K4me2 remained functional in the short term, but were defective in incorporation of CENP-A, and were gradually inactivated. Our data provide a functional link between the centromeric chromatin, α-satellite transcription, maintenance of CENP-A levels and kinetochore stability.

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.

Organization and function of the phage Lambda chromosome

The process of prophage integration by phage λ and the function and structure of the chromosomal elements required for λ integration have been studied with the use of λ deletion mutants. Since att^φ, the substrate of the integration enzymes, is not essential for λ growth, and since att^φ resides in a portion of the λ chromosome which is not necessary for vegetative growth, viable λ deletion mutants were isolated and examined to dissect the structure of att^φ.

Deletion mutants were selected from wild type populations by treating the phage under conditions where phage are inactivated at a rate dependent on the DNA content of the particles. A number of deletion mutants were obtained in this way, and many of these mutants proved to have defects in integration. These defects were defined by analyzing the properties of Int-promoted recombination in these att mutants.

The types of mutants found and their properties indicated that att^φ has three components: a cross-over point which is bordered on either side by recognition elements whose sequence is specifically required for normal integration. The interactions of the recognition elements in Int-promoted recombination between att mutants was examined and proved to be quite complex. In general, however, it appears that the λ integration system can function with a diverse array of mutant att sites.

The structure of att^φ was examined by comparing the genetic properties of various att mutants with their location in the λ chromosome. To map these mutants, the techniques of heteroduplex DNA formation and electron microscopy were employed. It was found that integration cross-overs occur at only one point in att^φ and that the recognition sequences that direct the integration enzymes to their site of action are quite small, less than 2000 nucleotides each. Furthermore, no base pair homology was detected between att^φ and its bacterial analog, att^B. This result clearly demonstrates that λ integration can occur between chromosomes which have little, if any, homology. In this respect, λ integration is unique as a system of recombination since most forms of generalized recombination require extensive base pair homology.

An additional study on the genetic and physical distances in the left arm of the λ genome was described. Here, a large number of conditional lethal nonsense mutants were isolated and mapped, and a genetic map of the entire left arm, comprising a total of 18 genes, was constructed. Four of these genes were discovered in this study. A series of λdg transducing phages was mapped by heteroduplex electron microscopy and the relationship between physical and genetic distances in the left arm was determined. The results indicate that recombination frequency in the left arm is an accurate reflection of physical distances, and moreover, there do not appear to be any undiscovered genes in this segment of the genome.

High-density comparative BAC mapping in the black muntjac (Muntiacus crinifrons): Molecular cytogenetic dissection of the origin of MCR 1p+4 in the X1X2Y1Y2Y3 sex chromosome system

The black muntjac (Muntiacus crinifrons, 2n = 8 female/9 male) is a critically endangered mammalian species that is confined to a narrow region of southeastern China. Male black muntjacs have an astonishing X1X2Y1Y2Y3 sex chromosome system, unparalleled i