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Molecular cloning and physical and genetic mapping of the human anion exchanger isoform 3 (SLC2C) gene to chromosome 2q36.

A human clone corresponding to the gene encoding anion exchanger isoform 3 (AE3) (approved gene symbol SLC2C) has been isolated and partially sequenced. Oligonucleotide primers based on this sequence were used in a polymerase chain reaction to specifically amplify a segment of the human gene from a panel of human-rodent somatic cell hybrids, allowing the assignment of AE3 to chromosome 2. To map AE3 more precisely to a cytogenetic band on chromosome 2, the AE3 cosmid was used as a probe in fluorescence in situ hybridization to human metaphase chromosomes. Fractional length measurements were made, and AE3 mapped at high resolution to the cytogenetic band 2q36. A polymorphic dinucleotide (GT/CA)n repeat marker was developed from sequences in the AE3 cosmid and typed on a subset of the CEPH families. Multipoint linkage analysis placed the AE3 gene between D2S128 and D2S126 on a genetic map of chromosome 2, corroborating the chromosomal localization of AE3 obtained by physical mapping methods.

Amino Acid Sequence↗

Fine genetic mapping of LCN1/D9S1826 within 9q34.

LCN1 gene encodes the tear lipocalin; the lipocalins are a large and growing family of proteins characterized by their ability to bind small hydrophobic molecules. We report here the location of a dinucleotide repeat microsatellite marker (D9S1826) close to LCN1 gene. Using the CEPH reference families, the position of LCN1 is located within the 9q34 genetic map between D9S23 and D9S158.

Carrier Proteins↗

Genetic mapping of the Kallmann syndrome and X linked ocular albinism gene loci.

The X linked form of Kallmann syndrome (KAL) and X linked ocular albinism (OA1) have both been mapped to Xp22.3. We have used a dinucleotide repeat polymorphism at the Kallmann locus to type 17 X linked ocular albinism families which had previously been typed for the Xg blood group (XG) and the DNA markers DXS237 (GMGX9), DXS143 (dic56), and DXS85 (782). Close linkage was found between KAL and OA1 with a maximum lod score (Zmax) of 30.14 at a recombination fraction (theta max) of 0.06 (confidence interval for theta: 0.03-0.10). KAL was also closely linked to DXS237 (Zmax = 15.32; theta max = 0.05; CI 0.02-0.12) and DXS143 (Zmax = 14.57; theta max = 0.05; CI 0.02-0.13). There was looser linkage to the Xg blood group (XG) and to DXS85 (782). Multipoint linkage analysis gave the map: Xpter-XG-0.13-DXS237-0.025-KAL-0.025-DXS143-0.01 5-OA1-0.09-DXS85-Xcen. Placement of OA1 proximal to DXS143 was supported by odds of 2300:1 compared to other orders. This confirms our previous localisation of OA1 and improves the genetic mapping of both disease loci.

Albinism↗

Rab17 and rab18, small GTPases with specificity for polarized epithelial cells: genetic mapping in the mouse.

The Rab subfamily of small GTPases plays an important role in the regulation of membrane traffic in eukaryotic cells. While most Rab proteins are equally expressed in polarized and nonpolarized cells, Rab17 and Rab18 show epithelial cell specificity. Here we report the genetic mapping of Rab17 and Rab18 on mouse chromosomes 1 and 18, respectively. We also discuss some implications of Rab17 and Rab18 mapping, including their candidacy for the mouse mutations ln (leaden), Tw (twirler), and ax (ataxia).

Animals↗

Genetic mapping and biochemical analysis of mutants in the maltose regulatory gene of the MAL1 locus of Saccharomyces cerevisiae.

The MAL1 locus of Saccharomyces cerevisiae comprises three genes necessary for maltose utilization: a regulatory (MALR), a maltose transport (MALT) and a maltase gene (MALS). A fine structure genetic map of the MAL1R gene was constructed and the order of mutations was confirmed by plasmid-mediated chromosomal recombination. The mutations cluster non-randomly within the 5' half of the gene, where the putative DNA binding domain of the encoded protein is located. Only mutations mal1R-22 and MAL1R-72 map in the 3' terminal half of the gene; these mutations cause a different pattern of transcriptional regulation of plasmid-borne MAL6T genes. Experiments supporting a direct involvement of the MALR-encoded protein in carbon catabolite repression of MAL gene expression are reported.

Blotting, Northern↗

Genetic mapping and transcriptional orientation of the fimD gene.

Expression of type 1 fimbriae in Escherichia coli exhibits phase variation whereby individual cells can alternate between states of organelle expression and nonexpression. Strains carrying fim-lac operon fusions in which lac operon expression is under the control of a fim promoter undergo Lac+ in equilibrium Lac- phase variation. We have determined the genetic map location and direction of transcription of a fim-lac operon fusion which was obtained by insertion of lac into a locus we have named fimD. We found the gene order to be as follows: valS fimD uxuA serB. The direction of transcription of fimD was found to be clockwise on the E. coli chromosome.

Chromosome Mapping↗

Genetic mapping of mutation in Escherichia coli leading to a temperature-sensitive RNase D.

In order to determine the metabolic role of RNase D in Escherichia coli, we have attempted to isolate strains deficient in this enzyme. One strain containing a temperature-sensitive RNase D was found among a heavily mutagenized stock of strain temperature-sensitive for growth. Genetic mapping of the mutation responsible for the altered RNAse D enabled us to define the rnd locus, at 39.5-40.0 min on the E. coli map, which apparently specifies the RNase D structural gene. Using a Tn10 insertion near the rnd locus, we constructed isogenic strains containing RNase D and Rnase II mutations, alone or in combination. Although the original mutant isolate displayed temperature-sensitive growth. no growth phenotype was associated with the rnd mutation in wild type background, possibly because a substantial amount of RNase D remained in cells grown at 45 degrees C. However, elucidation of the map position of the rnd locus should prove useful for the isolation of other mutant strains with lower levels of RNase D.

Alleles↗

Construction of a correlated physical and genetic map of the Klebsiella pneumoniae hisDGO region using transposon Tn5 mutagenesis.

Multicopy plasmids containing the hisDG region of Klebsiella pneumoniae were mutagenized with transposon Tn5. The resulting plasmids were examined for their ability to complement hisD and hisG mutations in Escherichia coli. The physical location of Tn5 on each of the hisD::Tn5 and hisG::Tn5 plasmids was determined by restriction endonuclease analysis. By combining the two types of data, a precise correlated physical and genetic map of the K. pneumoniae hisDG region was constructed. Based on this analysis, the minimum sizes of the hisD and the hisG genes were calculated to be 1100 bp and 900 bp, respectively. The hisO(P) region was also identified. The insertional specificity of transposon Tn5 was shown to be very low. One unanticipated result was obtained: Tn5 insertions in the plasmid-borne hisG gene were not polar on hisD.

Chromosome Mapping↗

Genetic mapping of ada and adc mutations affecting the adaptive response of Escherichia coli to alkylating agents.

The adaptive response to alkylating agents is an inducible repair system which protects Escherichia coli against the mutagenicity and toxicity of these agents. Four mutations, ada-3, ada-5, ada-6, and adc-1, which confer differing phenotypes as regards this response, were shown to be cotransducible with gyrA, and were located at 47 min on the E. coli genetic map. A mutation already shown on the map at 47 min as tag (B. J. Bachmann and K. B. Low, Microbiol. Rev. 44:1--56, 1980; Karran et al., J. Mol. Biol. 140:101--127, 1980) is now known to be an ada mutation (G. Evensen and E. Seeberg, personal communication).

Alkylating Agents↗

Physical and genetic mapping of the serpin gene cluster at 14q32.1: allelic association and a unique haplotype associated with alpha 1-antitrypsin deficiency.

The alpha 1-antitrypsin (PI) gene is part of a cluster of structurally related serine protease inhibitor genes localized at chromosome 14q32.1, a cluster that includes the alpha 1-antichymotrypsin (AACT), protein C inhibitor (PCI), and corticosteroid-binding globulin (CBG) genes and the alpha 1-antitrypsin-like pseudogene (PIL). The order of the genes is refined here by genetic mapping using simple tandem repeat polymorphisms (STRPs) and by physical mapping in YACs. The order of the genes is (centromere)-CBG-PIL-PI-PCI-AACT-(telomere). Analysis of DNA haplotypes comprising STRP and RFLP markers in the serpin genes reveals considerable allelic association throughout the cluster. Furthermore, the common alpha 1-antitrypsin deficiency allele, PI*Z, has a unique DNA haplotype at the CBG, PIL, and PI loci, which extends over 60 kb in 97% of cases and in 44% of cases includes the PCI and AACT loci. This unique haplotype will be of use in examining a number of other diseases, particularly those with an inflammatory component, thought to be associated with alpha 1-antitrypsin deficiency or partial deficiency.

Alleles↗

Constructing the parental linkage phase and the genetic map over distances <1 cM using pooled haploid DNA.

A new statistical approach for construction of the genetic linkage map and estimation of the parental linkage phase based on allele frequency data from pooled gametic (sperm or egg) samples is introduced. This method can be applied for estimation of recombination fractions (over distances <1 cM) and ordering of large numbers (even hundreds) of closely linked markers. This method should be extremely useful in species with a long generation interval and a large genome size such as in dairy cattle or in forest trees; the conifer species have haploid tissues available in megagametophytes. According to Mendelian expectation, two parental alleles should occur in gametes in 1:1 proportions, if segregation distortion does not occur. However, due to mere sampling variation, the observed proportions may deviate from their expected value in practice. These deviations and their dependence along the chromosome can provide information on the parental linkage phase and on the genetic linkage map. Usefulness of the method is illustrated with simulations. The role of segregation distortion as a source of these deviations is also discussed. The software implementing this method is freely available for research purposes from the authors.

Algorithms↗

Isolation and genetic mapping of a Coffea canephora phenylalanine ammonia-lyase gene (CcPAL1) and its involvement in the accumulation of caffeoyl quinic acids.

Biosynthesis of caffeoylquinic acids occurs via the phenylpropanoid pathway in which the phenylalanine ammonia-lyase (PAL) acts as a key-control enzyme. A full-length cDNA (pF6), corresponding to a PAL gene (CcPAL1), was isolated by screening a Coffea canephora fruit cDNA library and its corresponding genomic sequence was characterized. Amplification of total DNA from seven Coffea species revealed differences in intronic length. This interspecific polymorphism was used to locate the gene on a genetic map established for a backcross progeny between Coffea pseudozanguebariae and C. dewevrei. The CcPAL1 gene was found on the same linkage group, but genetically independent, as a caffeoyl-coenzyme A-O-methyltransferase gene, another gene intervening in the phenylpropanoid pathway. In the same backcross, a lower caffeoylquinic acid content was observed in seeds harvested from plants harbouring the C. pseudozanguebariae CcPAL1 allele. Involvement of the CcPAL1 allelic form in the differential accumulation of caffeoylquinic acids in coffee green beans is then discussed.

Amino Acid Sequence↗

Establishment of gene transfer systems for and construction of the genetic map of a marine Vibrio strain.

Two gene transfer systems were established for a marine bacterium, Vibrio sp. strain 60. One was generalized transduction with a newly isolated bacteriophage, As3, and the other was conjugal gene transfer by the use of newly constructed transposon-facilitated recombination (Tfr) donors. As3 transduced various chromosomal markers at frequencies of 10(-4) to 10(-6). Tfr donors, which were constructed by introducing transposon Tn10 into both plasmid RP4 and the chromosome, mediated the polarized transfer of chromosomal genes from the sites of Tn10 insertion on the chromosome. By means of these gene transfer systems, a genetic map of the vibrio chromosome was constructed.

Bacteriophages↗

Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG Gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis.

A 24 kilobase pair region of the E. coli chromosome surrounding the dnaG gene has been cloned and characterized. A lambda phage library was constructed by ligating a Sau3A( decreases GATC) partial DNA digest of the entire E. coli chromosome into the lambda BamHI(G decreases GATCC) cloning vector charon 28. Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome. This library was probed with a nick-translated plasmid (pRRBl) containing the rpoD gene, which maps adjacent to dnaG at 66 min. Four bacteriophages: lambda 3, lambda 4, lambda 5, lambda 6 that hybridized to the probe were isolated from the 2,500 plaques screened. One phage recombinant lambda 4, was shown to contain the dnaG gene. Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of lambda 4 using different restriction of fragments. Plasmids pGL444 and pBS10 5 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated. The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement of dnaGts chromosomal marker at nonpermissive 40 degrees C. In this manner a correlated physical and genetic map of dnaG was determined. A large number of Tn5 inserts map to a specific 900 b.p. region which we propose may be involved in the regulation of dnaG gene expression.

Bacterial Proteins↗

Macromolecular organization and genetic mapping of a rapidly evolving chromosome-specific tandem repeat family (B77) in cotton (Gossypium).

Isolation and characterization of the most prominent repetitive element families in the genome of tetraploid cotton (Gossypium barbadense L; [39]) revealed a small subset of families that showed very different properties in tetraploids than in their diploid progenitors, separated by 1-2 million years. One element, B77, was characterized in detail, and compared to the well-conserved 5S and 45S rRNA genes. The 572 bp B77 repeat was found to be concentrated in several discontinuous tandem arrays confined to a single 550 kb SalI fragment in tetraploid cotton. Genetic mapping based on the absence of the pentameric 'rung' in the G. barbadense 'ladder' showed that B77 maps to a D-subgenome chromosome. In situ hybridization supports the contention that the array is confined largely to a single chromosomal site in the D-subgenome. The B77 repeat has undergone a substantial increase in copy number since formation of tetraploid cotton from its diploid relatives. RFLPs observed among tetraploid cotton species suggest that amplification and/or rearrangement of the repeat may have continued after divergence of the five tetraploid cotton species. B77 contains many short direct repeats and shares significant DNA sequence homology with a Nicotiana alata retrotransposon Tna1-2 integrase motif. The recent amplification of B77 on linkage group D04 suggests that the D-subgenome of tetraploid cotton may be subject to different evolutionary constraints than the D-genome diploid chromosomes, which exhibit few genome-specific elements. Further, the abundance of B77 in G. gossypioides supports independent evidence that it may be the closest extant relative of the D-genome ancestor of cotton.

Base Sequence↗

Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome.

Spiroplasma kunkelii (class Mollicutes) is the characteristically helical, wall-less bacterium that causes corn stunt disease. A combination of restriction enzyme analysis, pulsed-field gel electrophoresis (PFGE), and Southern hybridization analysis was used to construct a physical and genetic map of the S. kunkelii CR2-3x chromosome. The order of restriction fragments on the map was determined by analyses of reciprocal endonuclease double digests employing I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI; adjacent fragments were identified on two-dimensional pulsed-field electrophoresis gels. The size of the chromosome was estimated at 1550 kb. Oligonucleotide pairs were designed to prime the amplification of 26 S. kunkelii gene sequences in the polymerase chain reaction (PCR). Using PCR amplicons as probes, the locations of 27 S. kunkelii putative single-copy genes were positioned on the map by Southern hybridization analyses of chromosomal fragments separated in PFGE. The nucleotide sequence of the single ribosomal RNA operon was determined and its location mapped to a chromosomal segment bearing recognition sites for SalI, SmaI, EagI, and I-CeuI.

Blotting, Southern↗

Mutation affecting thermostability of sigma subunit of Escherichia coli RNA polymerase lies near the dnaG locus at about 66 min on the E. coli genetic map.

The Escherichia coli strain, ts-rnp5, originally described in 1975 by G. D. Burdick and H. Berger, is shown to possess an RNA polymerase (RNA nucleotidyltransferase) sigma subunit with an activity 4--6 times less thermostable at 45 degrees than sigma from wild-type strains. This defect remains associated with the sigma polypeptide through a variety of purification stages, including renaturation of sigma after its elution from sodium dodecyl sulfate/polyacrylamide gels. The mutation responsible for decreased thermostability of sigma, called rpoD1, cotransduces with dnaG and therefore is located at about 66 min of the E. coli genetic map.

Chromosome Mapping↗

Physical map of the Salmonella typhimurium histidine transport operon: correlation with the genetic map.

A detailed restriction map of a 12.4-kilobase EcoRI fragment of Salmonella typhimurium deoxyribonucleic acid (DNA) containing the entire histidine transport operon and the argT gene is presented. Subclones of specific regions of the transport operon of S. typhimurium were constructed in plasmid vectors. An accurate correlation between the restriction map and the location of genetically defined deletions was obtained by hybridizing restriction digests of chromosomal DNA from strains carrying each deletion with cloned transport operon DNA as a probe. These data were used to position the histidine transport genes on the cloned 12.4-kilobase fragment of DNA.

Biological Transport↗