A Diphenol Oxidase Gene Is Part of a Cluster of Genes Involved in Catecholamine Metabolism and Sclerotization in Drosophila. II. Molecular Localization of the Dox-A2 Coding Region

Mutations at the Dox-A2 (2-53.9) locus alter the A2 component of diphenol oxidase, an enzyme having an important role in cuticle formation. This locus is in the dopa decarboxylase, Df(2L)TW130 region, which contains a cluster of at least 14 genes involved in catecholamine metabolism and the formation, sclerotization and melanization of cuticle in Drosophila. The region is subdivided by deficiencies, and localization of breakpoints in cloned DNA reveals a dense subcluster of six genes in the 23 kb proximal to Ddc. Five lethal loci distal to Ddc comprise a second such subcluster. The proximal breakpoints of deficiencies Df(2L)hk18 and Df(2L)OD15 define a 14.3- to 16.8-kb region containing Dox-A2 and l(2)37Bb, and those of Df(2L)OD15 and Df(2L)TW203 define a 9.3- to 12.1-kb region containing l(2)37Ba, l(2)37Bc and l(2)37Be. Southern blots show two of the Dox-A2 mutations are small deletions (0.1 and 1.1 kb). The Dox-A2 locus mRNA is 1.7 kb. cDNA clones indicate that the 3' end is centromere proximal and that the coding region contains at least one small intron. The Dox-A2 locus is within 3.4 to 4.4 kb of the Df(2L)OD15 breakpoint, placing four of the vital loci within a maximum of 15.5 kb. The location of Dox-A2 in a cluster of genes affecting cuticle formation is discussed.     

The molecular analyses of an antimorphic mutation of Drosophila melanogaster, Scutoid

A dominant mutation of Drosophila melanogaster, Scutoid (Sco), acts as an antimorphic allele of the no-ocelli (noc) gene. In Sco the noc region has been transposed from 35B to 35D on chromosome arm 2L and the noc gene is now adjacent to snail (sna). Induced revertants of Sco are frequently mutant for sna or are aberrations broken very close to sna. A molecular analysis of the Sco chromosome has confirmed that noc is transposed and fused to the sna region. However, only part of the noc region is included within the transposition. The breakpoints of 19 chromosomally aberrant Sco revertants have been mapped at the molecular level. Fourteen of these breakpoints map to the noc region, spread over about 80 kb of DNA. The breakpoints of the remaining five are not within the DNA of the noc region and appear to map within sequences from the sna region. This has been shown directly for three of these, those associated with T(2;3)ScoR+13, In(2L)ScoR+24 and In(2L)ScoR+26. Thus mutation of either noc or sna, genes which are apparently unrelated in their wild-type functions, can revert the antimorphic phenotype of Sco.     

A bacterial artificial chromosome library for 'Jefferson' hazelnut and identification of clones associated with eastern filbert blight resistance and pollen-stigma incompatibility

European hazelnut (Corylus avellana L.) is the only economically important nut crop in the family Betulaceae. Because of its small genome size (~385 Mb / 1C), relatively short life cycle, availability of a dense linkage map, and amenability to transformation by Agrobacterium, the European hazelnut could serve as a model plant for the Betulaceae. Here we report the construction of a bacterial artificial chromosome (BAC) library for 'Jefferson' hazelnut using the cloning enzyme MboI and the vector pECBAC1 (BamHI site). The library consists of 39,936 clones arrayed in 104,384-well microtitre plates with a mean insert size of 117 kb. The genomic coverage of the library is estimated to be about 12 genome equivalents. This library provides a valuable resource for the map-based cloning of two important genes, the resistance gene from 'Gasaway' that confers resistance to eastern filbert blight caused by the fungus Anisogramma anomala (Peck) E. Müller and the S locus that controls pollen-stigma incompatibility. Fine-resolution mapping near the two loci was carried out using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. Fine mapping at the disease resistance locus showed that markers W07-375 and X01-825 flanked the resistance locus at distances of 0.06 and 0.05 cM, respectively. The S locus is flanked by markers 204-950 and KG819-200 at distances of 0.14 and 0.24 cM, respectively. Assuming that 1 cM corresponds to a physical distance of 430 kb, it will take approximately two to three chromosome walks to assemble BAC contigs that span both loci.     

Molecular analysis of the Adh region of the genome of Drosophila melanogaster

A small region of the genome of Drosophila melanogaster has been cloned in a series of overlapping phage. A length of 165 X 10(3) base-pairs of contiguous DNA that spans polytene chromosome region 35A4 to 35B1 and includes the structural gene for alcohol dehydrogenase (Adh) as well as at least two other genes, outspread (osp) and no-ocelli (noc), has been characterized by mapping chromosome aberrations to the DNA. The relationship between osp and Adh is surprising: of nine osp alleles associated with chromosome breakpoints, five map distal (i.e. 5') to Adh and four map proximal (i.e. 3') to this gene. None affects the expression of Adh. As defined by these and other breakpoints, the osp gene spans at least 52 X 10(3) base-pairs and overlaps the Adh gene. The noc gene, as defined by the mapping of nearly 30 breakpoints, is at least 50 X 10(3) base-pairs in size. Alleles of noc and noc- deletions show either of two kinds of interaction with the recessive lethality of l(2)br29ScoR+1, a lethal that maps immediately distal to noc. One class of noc allele is viable when heterozygous with ScoR+1, while the other class is lethal or semi-lethal. Both classes, however, are homozygous or hemizygous viable. The locations of these two classes of noc allele on the DNA fall into two clusters, with those that are viable with ScoR+1 located proximal to those that are not. The physical boundary between these classes lies at a site just distal to that of the breakpoint of the inversion associated with ScoR+1 itself.     

Mapping and characterization of the DQ subregion of the ovine MHC

A map of the ovine MHC class II DQ subregion has been constructed from overlapping cosmid clones. This region consists of two loci linked on a linear tract of 130 kb DNA. Each locus consists of a DQA and a DQB gene in a tail-to-tail orientation. The genes in each locus are transcribed but only those designated DQ1 express class II molecules at the surface of mouse L cells following DNA-mediated gene transfection. The DQA1 and DQB1 genes are separated by 11kb while the DQA2 and B2 genes are 25 kb apart. The loci are separated by 22 kb.     

Physical mapping of four serpin genes: alpha 1-antitrypsin, alpha 1-antichymotrypsin, corticosteroid-binding globulin, and protein C inhibitor, within a 280-kb region on chromosome I4q32.1.

Alpha 1-antitrypsin (alpha 1AT; protease inhibitor [PI] locus), alpha 1-antichymotrypsin (alpha 1ACT; AACT locus), corticosteroid-binding globulin (CBG; CBG locus), and protein C inhibitor (PCI; PCI locus) are members of the serine protease inhibitor (serpin) superfamily. A noncoding PI-like (PIL) gene has been located 12 kb 3' of the PI gene. The PI, PIL, and AACT loci have been localized to 14q32.1, the CBG locus has been localized to 14q31-14q32.1, and PCI has been mapped to chromosome 14. Genetic linkage analysis suggests tight linkage between PI and AACT. We have used pulsed-field gel electrophoresis to generate a physical map linking these five serpin genes. The order of the genetic loci is AACT/PCI-PI-PIL-CBG, with a maximum distance of about 220 kb between the AACT/PCI and PI genes. These genes form a PI cluster at 14q32.1, similar to that of the homologous genes on murine chromosome l2. The close proximity of these genes has implications for disease-association studies.     

Construction of an RFLP map in sorghum and comparative mapping in maize.

An F2 population derived from a cross between Sorghum bicolor ssp. bicolor ('CK60') and Sorghum bicolor ssp. drummondii ('PI229828') was used to develop an RFLP genetic linkage map of sorghum. The map consists of 201 loci distributed among 10 linkage groups covering a map distance of 1530 cM, with an average 8 cM between adjacent loci. Maize genomic probes (52), maize cDNA probes (124), and sorghum genomic probes (10) were used to define the loci (55, 136, and 10, respectively). Ninety-five percent of the loci fit expected segregation ratios. The loci with distorted segregation ratios were confined almost exclusively to a region of one linkage group. Comparison of sorghum and maize maps indicated high correspondence between the two genomes in terms of loci order and genetic distance. Many loci linked in maize (45 of 55) were also linked in sorghum. Instances of both conserved and rearranged locus orders were detected.     

Molecular genetic analysis of theripening-inhibitor andnon-ripening loci of tomato: A first step in genetic map-based cloning of fruit ripening genes

Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato,ripening-inhibitor (rin), andnon-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning therin andnor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning therin locus is estimated to be 200–300 kb/cM, while thenor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked torin andnor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains thenot locus.     

Molecular genetic analysis of theripening-inhibitor andnon-ripening loci of tomato: A first step in genetic map-based cloning of fruit ripening genes

Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato,ripening-inhibitor (rin), andnon-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning therin andnor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning therin locus is estimated to be 200–300 kb/cM, while thenor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked torin andnor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains thenot locus.     

A new family of polymorphic metallothionein-encoding genes MTH1 (CUP1) and MTH2 in Saccharomyces cerevisiae.

By pulsed-field gel electrophoresis of chromosomal DNA and hybridization with a cloned MTH1 (CUP1) gene, we determined the locations of metallothionein-encoding gene sequences on chromosomes in monosporic cultures of 76 natural strains of Saccharomyces cerevisiae. Most of the strains (68) exhibited a previously known location for the MTH sequence on chromosome (chr.) VIII. Seven strains (resistant or sensitive to Cu2+) showed a MTH sequence in a new locus, MTH2, on chr. XVI. One strain carried an MTH locus on both chromosomes VIII and XVI. Restriction fragment and Southern blot analyses showed that the two MTH loci were very closely related. The strains displayed heterogeneity in the size and structure of their MTH2 locus. The length of the repeat unit of MTH2 varied: a 1.9-kb or 1.7-kb unit was found, instead of the 2.0-kb unit of the MTH1 locus. The most resistant strain (resistant to 1.2 mM CuSO4) contained a 0.9-kb repeat unit in addition to those of 1.9 kb and 1.7 kb. All three sensitive (to over 0.3 mM CuSO4) strains with an mth2 locus had a repeat unit of 1.9 kb or 1.7 kb, suggesting the presence of at least two copies of the MTH2 gene, with one always being in the junction area outside of the repeat unit. A monogenic tetrad segregation of 2:2 was usually found in crosses of resistant MTH2 and sensitive mth2 strains. Hybrids between strains with different MTH loci in all combinations showed low ascospore viability, suggesting that the complete lack of an MTH locus may lead to the death of segregants on YPD medium. The MTH1 and MTH2 loci were exchangeable. Strains with a high level of Cu2+ resistance were also resistant to Cd2+. However, these two properties did not cosegregate in heterozygotic hybrids.     

Growth hormone locus expands and diverges after the separation of New and Old World Monkeys

While most mammals including the prosimians have a single copy of the growth hormone (GH) gene, anthropoids possess a cluster of GH-related genes. Throughout the evolution of the main anthropoid groups [New World Monkeys (NWM), Old World Monkeys (OWM), and apes], two features stand out of the GH loci. The first is the appearance of chorionic somatommamotropin hormone (CSH) genes within the OWM lineage and the second is the expansion of the loci intergenic regions in the OWM and apes. In relation with this loci expansion, the NWM possess intergenic regions of homogeneous lengths (3.5 kb). In contrast, heterogeneous lengths (6 and 13 kb) have been reported for species of the OWM. At the present, none of the OWM genomic GH loci organizations have been described. Here, we report the genomic organization of the GH locus in the rhesus monkey, this locus has six GH-related genes separated by five intergenic regions. The 5' end gene (GH-1) encodes for the pituitary GH and is followed by CSH-1, GH-2, CSH-2, CSH-3 and CSH-4 genes. The five intergenic regions have heterogeneous lengths and also present more or less the same Alu distribution as the human GH locus. To analyze the events that contributed to the extension of the intergenic regions of the GH locus and the emergence of the regulatory elements, the five GH locus intergenic regions of the spider monkey (NWM) were sequenced. The results of comparing the loci from both species suggest that the long intergenic regions (13 kb) of the rhesus GH locus share a common ancestor with the 3.5 kb intergenic regions of the spider monkey. However, the observed increased length of the former is due to an insertion (approximately 8.7 kb) at their 3' end. Interestingly in this insert, we discovered a DNA element resembling the enhancer of the CSH genes of the human GH locus. On the other hand, we observed that the short intergenic regions (6 kb) increased by a different recombination event.     

High-resolution quantitative trait locus analysis reveals multiple diabetes susceptibility loci mapped to intervals<800 kb in the species-conserved Niddm1i of the GK rat

Niddm1i, a 16-Mb locus within the major diabetes QTL in the diabetic GK rat, causes impaired glucose tolerance in the congenic NIDDM1I strain. Niddm1i is homologous to both human and mouse regions linked with type 2 diabetes susceptibility. We employed multiple QTL analyses of congenic F2 progeny selected for one recombination event within Niddm1i combined with characterization of subcongenic strains. Fine mapping located one hyperglycemia locus within 700 kb (Niddm1i4, P=5x10(-6)). Two adjacent loci were also detected, and the GK allele at Niddm1i2 (500 kb) showed a glucose-raising effect, whereas it had a glucose-lowering effect at Niddm1i3 (400 kb). Most proximally, Niddm1i1 (800 kb) affecting body weight was identified. Experimental data from subcongenics supported the four loci. Sorcs1, one of the two known diabetes susceptibility genes in the region, resides within Niddm1i3, while Tcf7l2 maps outside all four loci. Multiple-marker QTL analysis incorporating the effect of cosegregating QTL as cofactors together with genetically selected progeny can remarkably enhance resolution of QTL. The data demonstrate that the species-conserved Niddm1i is a composite of at least four QTL affecting type 2 diabetes susceptibility and that two adjacent QTL (Niddm1i2GK and Niddm1i3GK) act in opposite directions.     

Pulsed-field gel electrophoresis analysis of the phaseolin locus region in Phaseolus vulgaris.

Phaseolin is the major seed storage protein of common bean (Phaseolus vulgaris L.). It is encoded by a small multigene family of 6-9 genes that are clustered in a single complex locus (Phs). We have constructed a long-range restriction map of the phaseolin genomic region, including the Phs locus and two flanking marker loci, D1861 and Bng060. Using a combination of high molecular weight DNA isolation, one- and two-dimensional pulsed-field gel electrophoresis of single and double restriction digests followed by Southern hybridization, and PCR analysis of individual fragments, we found that: (i) the maximum size of the Phs locus is 190 kb, (ii) the Phs locus may have increased in size during the evolution of P. vulgaris, (iii) the genomic region marked by D1861-Phs-Bng060 spans 5 cM, which corresponds to a maximum of 1.9 Mb, and (iv) the Phs locus could be oriented with respect to the two adjacent markers. Further progress in determining the gene arrangement in the Phs locus will require cloning and analysis of large DNA fragments containing phaseolin genes via BAC libraries. Key words : multigene family, physical distance, genome mapping, seed protein.     

Chromosomal arrangement of leghemoglobin genes in soybean.

A cluster of four different leghemoglobin (Lb) genes was isolated from AluI-HaeIII and EcoRI genomic libraries of soybean in a set of overlapping clones which together include 45 kilobases (kb) of contiguous DNA. These four genes, including a pseudogene, are present in the same orientation and are arranged in the order: 5'-Lba-Lbc1-Lb psi-Lbc3-3'. The intergenic regions average 2.5 kb. In addition to this main Lb locus, there are other Lb genes which do not appear to be contiguous to this locus. A sequence probably common to the 3' region of Lb loci was found flanking the Lbc3 gene. The 3' flanking region of the main Lb locus also contains a sequence that appears to be expressed more abundantly in root tissue. Another sequence which is primarily expressed in root and leaf is found 5' to two Lb loci. Overall, the main leghemoglobin locus is similar in structure to the mammalian globin gene loci.     

The murine genes Hox-5.1 and Hox-4.1 belong to the same HOX complex on chromosome 2

Two different loci of Antennapedia-related homeobox-containing genes have been shown to map to mouse chromosome 2: the HOX-5 complex and the Hox-4.1 gene. These independently derived loci are likely to be parts of a single gene complex, although their close linkage has not yet been demonstrated. Since cosmid walks to extend the HOX-5 cluster and to potentially link the two loci were unsuccessful, we have used large restriction fragments separated by pulsed-field gel electrophoresis to demonstrate the linkage between probes from the HOX-5 region and sequences near Hox-4.1. To further define the distance between the two linked loci, we screened a NotI jumping library with sequences near the Hox-5.1 gene to obtain a marker within the region predicted to contain Hox-4.1. The jumping endpoint lies within genomic clones from a lambda phage walk extending from the 5' end of Hox-4.1, and thus provides clear evidence of linkage between the two Hox loci. Our results demonstrate that Hox-4.1 lies approximately 35 kb downstream of the Hox-5.1 gene and that the two loci do indeed thus constitute parts of the same HOX complex.     

Evolution of chorion gene families in Lepidoptera: Characterization of 15 cDNAs from the gypsy moth

Fifteen unique chorion protein-encoding cDNAs from gypsy moth have been completely sequenced. These sequences are encoded by a family of genes, based on pairwise similarity values of 78–100% within a 225-nt region. Pairwise comparisons and maximum parsimony analysis strongly support the existence of two clusters of 11 and four sequences each, called noel and noc2. While noc2 consists of two subclusters, there is little character support for subclusters within noel. The highly localized character-state distribution on the parsimony tree in gypsy moth is reminiscent of that in Bombyx mori, specifically for those chorion families that have been shown to undergo gene conversion. Gene conversion thus becomes a reasonable explanation for the homogeneity of noel sequences and for their distinctness from noc2. The relationship between the two major clusters of chorion sequences in gypsy moth (noel, noc2) and Bombyx mori (Bm , Bm ) has been addressed through mixed-species tree construction. All four groups cluster separately, thus providing no direct evidence of orthologous sequences. However, the occurrence of gene conversion could have eliminated such evidence. The relationship between the chorion gene tree and the species cladogenic event is discussed, as are biases in codon usage, base composition, and nucleotide transformations.Correspondence to: J.C. Regier