Microdissection and microcloning of the long arm of human chromosome 7

DNA-fragments from the region of the long arm of human chromosme 7 to which the CF-locus has been mapped recently were isolated by microdissection and microcloning. We developed a new fixation procedure resulting in inserts of 1.0–7.0 kb in length with a mean value of 2.9 kb. Regional mapping of three clones on 7q was carried out by the use of different hybrid cell lines containing fragments of human chromosome 7.     

Microdissection of and microcloning from the short arm of human chromosome 2.

A bank of cloned DNA sequences from the distal half of the short arm of human chromosome 2 was generated by using microdissection and microcloning techniques. DNA was purified from 106 chromosomal fragments, manually dissected from peripheral lymphocytes in metaphase, and cloned into the EcoRI site of lambda gt10. A total of 257 putative recombinants were recovered, of which 41% were found to contain human inserts. The mean insert size was 380 base pairs (median size, 83 base pairs), and fewer than 10% of the clones contained highly repetitive sequences. All single-copy sequences examined were shown to map to the short arm of chromosome 2 by using hybrid panels. This technique provides a rapid method of isolating probes specific to a human subchromosomal region to generate linked markers to genetic diseases for which the chromosomal location is known.     

Microdissection of proximal mouse Chromosome 6: identification of RFLPs tightly linked to the ob mutation

In a previous report, the ob mutation was mapped to a position 5 cM distal to Met on murine Chromosome (Chr) 6 in tight linkage to Cpa. In order to identify additional RFLPs in the region of ob, we have made use of chromosome microdissection of a 6:16 Robertsonian chromosome. In total, 19 RFLPs were used to type 131 progeny of a B6D2 ob/+xB6 spretus ob/+intercross. Fifteen of the RFLPs mapped to Chr 6, one of which, D6Rck13, was tightly linked to ob. For refinement of the genetic map around ob, 350 obese progeny of a B6 Mus castaneus ob/+ intercross were characterized. DNAs from these animals were typed for microsatellite markers from Chr 6 that flank ob. Recombinants were then typed for D6 Rck13. D6Rck13 was nonrecombinant among all the progeny of both crosses corresponding to 831 meioses. This probe will be of use as an entry point for physical mapping of the ob mutation.     

Differential inspiratory timing is genetically linked to mouse chromosome 3

Genetic control ofdifferential inspiratory timing(TI) at baseline has beenpreviously demonstrated among inbred mouse strains. The inheritancepattern for TI between C3H/HeJ(C3; 188 ± 3 ms) and C57BL/6J (B6; 111 ± 2 ms) progenitors wasconsistent with a two-gene model. By using the strain distributionpattern for recombinant inbred strains derived from C3 and B6progenitors, 100% concordance was established betweenTI phenotypes and DNA markers onmouse chromosome 3. This genotype-phenotype hypothesis was tested bytyping 52 B6C3F₂(F₂) progeny by using simplesequence repeat DNA markers (n = 21)polymorphic between C3 and B6 strains on mouse chromosome 3. Linkageanalysis compared marker genotypes to baseline ventilatory phenotypesby computing log-likelihood values. A putative quantitative trait locuslocated in proximity to D3Mit119 wassignificantly associated with baselineTI phenotypes. At the peak(log-likelihood = 3.3), the putative quantitative trait locusdetermined 25% of the phenotypic variance inTI among F₂ progeny. In conclusion, thisgenetic model of ventilatory characteristics demonstrated an importantlinkage between differential baseline TI and a candidate genomicregion on mouse chromosome 3.

     

Microdissection and microcloning of the barley (Hordeum vulgare L.) chromosome 1HS

We have applied a refined microdissection procedure to create a plasmid library of the barley (Hordeum vulgare L.) chromosome arm 1HS. The technical improvements involved include synchronization of meristematic root tissue, a metaphase drop-spread technique, paraffin protection of the collection drop to avoid evaporation, and a motorized and programmable microscope stage. Thirteen readily-discernible telocentric chromosomes have been excised from metaphases of synchronized root-tip mitoses. After lysis in a collection drop (2 nl), the DNA was purified, restricted withRsaI, ligated into a vector containing universal sequencing primers, and amplified by the polymerase chain reaction. Finally, the amplified DNA was cloned into a standard plasmid vector. The size of the library was estimated to be approximately 44,000 recombinant plasmids, of which approximately 13% can be utilized for RFLP analysis. Tandem repetitive probes could be rapidly excluded from further analysis after colony hybridization with labelled total barley DNA. Analysis of 552 recombinant plasmids established that: (1) the insert sizes ranged between 70 and 1150 bp with a mean of 250 bp, (2) approximately 60% of the clones contained highly repetitive sequences, and (3) all single- or low-copy probes tested originate from chromosome 1HS. Four probes were genetically mapped, using an interspecificH. vulgare xH. spontaneum F₂ population. One of these probes was found to be closely linked to theMla locus conferring mildew resistance.     

Microdissection and microcloning of rye (Secale cereale L.) chromosome 1R

Chromosome 1R was microdissected and collected from mitotic metaphase spreads of rye (Secale cereale L.) by using glass needles. The isolated chromosomes were amplified in vitro by Sau3A linker adaptor-mediated polymerase chain reaction (PCR). After amplification, the presence of rye-specific DNA was verified by Southern hybridization. The second-round PCR products from five 1R chromosomes were cloned into a plasmid vector to create a chromosome-specific library, which produced approximately 220,000 recombinant clones. Characterization of the microclone library showed that the 172 clones evaluated ranged in size from 300–1800 bp with an average size of 950 bp, of which approximately 42% were medium/high copy and 58% were low/unique copy clones. Chromosome in situ hybridization confirmed that the PCR products from microdissected chromosomes originated from chromosome 1R, indicating that many chromosome 1R-specific sequences were present in the library. Received: 5 December 1998; in revised form: 15 April 1999 / Accepted: 29 April 1999     

Erbb is linked to the alpha-globin locus on mouse chromosome 11.

A fragment of the human gene for c-erb-B was used to map homologous sequences in mice. Analysis of somatic cell hybrids and recombinant inbred and congenic mouse strains indicated that this gene, designated Erbb, is closely linked to the gene for alpha-globin on mouse chromosome 11. Several genes controlling hematopoietic differentiation map to mouse chromosome 11.     

The syntenic relationship of proximal mouse chromosome 7 and the myotonic dystrophy gene region on human chromosome 19q

The syntenic relationship of the myotonic dystrophy (DM) gene region on human chromosome 19q and proximal mouse chromosome 7 was examined using an interspecific backcross between C3H/HeJ-gld/gld mice and Mus spretus. Segregation analyses were used to order homologs of nine human loci linked with the DM gene. Their order from the centromere was Prkcg, [Apoe, Atpa-2, Ckmm, D19S19h, Ercc-2], Cyp2b, Mag, Lhb. Two other murine loci, D7Rp2 and Ngfg, were also positioned within this interval. Homologs for five human chromosome 11 and 15 loci (Calc, Fes, Hras-1, Igflr, Tyr) were localized within an 18-cM span telomeric to Lhb. Comparison of the gene orders indicates an inversion extending from Prkcg through the interval between Mag and Lhb. This study establishes a detailed map of proximal mouse chromosome 7 that will be useful in identifying and determining whether new human chromosome 19 probes are linked to the DM region.     

The mouse uracil-DNA glycosylase gene: isolation of cDNA and genomic clones and mapping ung to mouse chromosome 5

Uracil-DNA glycosylase (UDG) is the enzyme responsible for the first step in the base-excision repair pathway that specifically removes uracil from DNA. Here we report the isolation of the cDNA and genomic clones for the mouse uracil-DNA glycosylase gene (ung) homologous to the major placental uracil-DNA glycosylase gene (UNG) of humans. The complete characterization of the genomic organization of the mouse uracil-DNA glycosylase gene shows that the entire mRNA coding region for the 1.83-kb cDNA of the mouse ung gene is contained in an 8.2-kb SstI genomic fragment which includes six exons and five introns. The cDNA encodes a predicted uracil-DNA glycosylase (UDG) protein of 295 amino acids (33 kDa) that is highly similar to a group of UDGs that have been isolated from a wide variety of organisms. The mouse ung gene has been mapped to mouse chromosome 5 using fluorescence in situ hybridization (FISH).     

Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region

To isolate genes of interest in plants, it is essential to construct bacterial artificial chromosome (BAC) libraries from specific genotypes. Construction and organisation of BAC libraries is laborious and costly, especially from organisms with large and complex genomes. In the present study, we developed the pooled BAC library strategy that allows rapid and low cost generation and screening of genomic libraries from any genotype of interest. The BAC library is constructed, directly organised into a few pools and screened for BAC clones of interest using PCR and hybridisation steps, without requiring organization into individual clones. As a proof of concept, a pooled BAC library of approximately 177,000 recombinant clones has been constructed from the barley cultivar Cebada Capa that carries the Rph7 leaf rust resistance gene. The library has an average insert size of 140 kb, a coverage of six barley genome equivalents and is organised in 138 pools of about 1,300 clones each. We rapidly established a single contig of six BAC clones spanning 230 kb at the Rph7 locus on chromosome 3HS. The described low-cost cloning strategy is fast and will greatly facilitate direct targeting of genes and large-scale intra- and inter-species comparative genome analysis.Edwige Isidore and Beatrice Scherrer contributed equally to the work.     

Localization of two alkaline phosphatase genes to the proximal region of mouse chromosome 1

Using a human cDNA clone encoding the intestinal form of alkaline phosphatase, we have identified and mapped by RFLP analysis in a Mus spretus x C57BL/6J interspecies backcross two alkaline phosphatase genes which segregate independently on the proximal part of mouse Chromosome 1. The gene order and intergene distances were determined by standard backcross analysis as: centromere- Len-2 - 19.0 cM - Akp-3 - 20.0 cM - Akp-4 - 2.0 cM - Ren-1.     

Construction of a rice bacterial artificial chromosome library and identification of clones linked to the Xa-21 disease resistance locus

A bacterial artificial chromosome (BAC) library consisting of 11 000 clones with an average DNA insert size of 125 kb was constructed from rice nuclear DNA. The BAC clones were stable in E. coli after 100 generations of serial growth. Transformation of the BAC clones by electroporation into E. coli was highly efficient and increased with decreasing size of the DNA inserts. The library was evaluated for the presence of organellar, repeated, and telomeric sequences. A very low percentage (<0.3%) of the library consisted of chloroplast and mitochondrial clones. Eighteen BACs were identified that hybridized with an Arabidopsis telomere repeat. Sixteen BACs hybridized with the AA genome-specific repetitive sequence pOs48. Twelve clones were isolated that hybridized with three DNA markers linked to the Xa-21 disease resistance locus. The results indicate that the BAC system can be used to clone and manipulate large pieces of plant DNA efficiently.     

Establishment of the mouse chromosome 7 region with homology to the myotonic dystrophy region of human chromosome 19q

A number of genetic markers, including ATP1A3, TGFB, CKMM, and PRKCG, define the genetic region on human chromosome 19 containing the myotonic dystrophy locus. These and a number of other DNA probes have been mapped to mouse chromosome 7 utilizing a mouse Mus domesticus/Mus spretus interspecific backcross segregating for the genetic markers pink-eye dilution (p) and chinchilla (cch). The establishment of a highly syntenic group conserved between mouse chromosome 7 and human chromosome 19q indicates the likely position of the homologous gene locus to the human myotonic dystrophy gene on proximal mouse chromosome 7. In addition, we have mapped the muscle ryanodine receptor gene (Ryr) to mouse chromosome 7 and demonstrated its close linkage to the Atpa-2, Tgfb-1, and Ckmm cluster of genes. In humans, the malignant hyperthermia susceptibility locus (MHS) also maps close to this gene cluster. The comparative mapping data support Ryr as a candidate gene for MHS.     

In situ hybridization of two cloned chromosome 7 sequences tightly linked to the cystic fibrosis locus

Two DNA sequences closely linked to the cystic fibrosis locus have been sublocalized to 7q31.3----q32 by in situ hybridization. These findings are consistent with previously published maps of that region of human chromosome 7. The cystic fibrosis locus therefore maps to the 7q31.3----q32 region, a more distal location that had been inferred from previous data.     

Mapping of DNA markers linked to the cystic fibrosis locus on the long arm of chromosome 7.

We have used a panel of eight human/mouse somatic-cell hybrids, each containing various portions of human chromosome 7, and three patient cell lines with interstitial deletions on chromosome 7 for localization of six DNA markers linked to the cystic fibrosis locus. Our data suggest that D7S15 is located in the region 7 cen----q22, that MET is located in 7q22----31, and that D7S8 and 7C22 are located in q22----q32. The hybridization results for COL1A2 and TCRB are consistent with their previous assignment to 7q21----q22 and 7q32, respectively. Given the location of these six markers and their linkage relationships, it is probable that the cystic fibrosis locus is in either the distal region of band q22 or the proximal region of q31. Using the same set of cell lines, we have also examined the location of another chromosome 7 marker PGY1. The data show that PGY1 is located in the region 7cen----q22, a position very different from its previous assignment.     

CD19 maps to a region of conservation between human chromosome 16 and mouse chromosome 7

CD19 is a B lymphocyte cell surface protein expressed from the earliest stages of B lymphocyte development unitl their terminal differentiation into plasma cells. In this report the human CD19 gene (hCD19) was localized to band p11.2 on the proximal short arm of chromosome 16 by in situ hybridization to metaphase chromosomes, using hCD19 cDNA as probe. hCD19 gene localization was confirmed by polymerase chain reaction based analysis with hCD19-specific primers, using a panel of human/hamster somatic cell hybrid DNA as templates. The mouse CD19 gene (MCd19) was mapped to bands F3-F4 of chromosome 7 by in situ hybridization to metaphase chromosomes, using a mCD19 cDNA probe. Segregation analysis of nucleotide sequence polymorphisms in inter-specific backcross progeny revealed linkage of mCd19 with hemoglobin (Hbb), Int-2, and H19, other loci previously mapped to the same region of mouse chromosome 7, confirming the localization of mCd19 to this region. The order of these loci was determined to be centromere — Hbb — mCd19 — H19 — Int-2 —telomere. The genetic distance between the loci examined, calculated from the recombination frequencies, suggested that mCd19 was located centrally between Hbb and H19. This region of mouse chromosome 7 is homologous to the region of human chromosome 16 to which the hCD19 gene maps. Multiple genes with a lymphocyte-related function also map to this conserved region including genes encoding the IL-4 receptor, CD11a, CD11b, CD11c, CD43 (leukosialin), and protein kinase C polypeptide.