Restriction Fragment Length Variants Research Articles

Evolutionary conservation of the mouse apolipoprotein e-c1-c2 gene cluster: structure and genetic variability in inbred mice.

The human apolipoprotein E (APOE), APOC1, pseudo APOC1 (APOC1'), and APOC2 genes are clustered within 48 kb on the long arm of chromosome 19. A mouse Apoe cDNA probe was used to isolate overlapping cosmid clones from a cosmid library of the C57BL/Rij inbred mouse strain. These clones were investigated for the presence of the Apoc1 and Apoc2 genes by heterologous hybridization. Our results show that the Apoe-c1-c2 gene cluster is conserved in the mouse. In line with evolutionary data, the mouse lacks the equivalent of APOC1'. These data were confirmed using a mouse Apoc2 cDNA clone, and surprisingly the cDNA clone isolated here was 965 bp in size, which is on average 450 bp longer than other APOC2 cDNAs described so far. Correspondingly, the Apoc2 gene occupies an unusually large genomic region, due to an extended 5' end. Interestingly, a variable number of tandem repeat (VNTR) in the third intron of the human APOC2 gene shows a high sequence homology and is located at the identical position in the mouse gene. Despite the high copy number of this VNTR (27 or 34 copies) only two variants were found among 11 different inbred strains. With the aid of six restriction fragment length variations in this gene cluster only two different haplotypes could be deduced, indicating that the Apoe-c1-c2 gene cluster is highly conserved in the inbred strains that were studied.

Chromosomal assignment of the recoverin gene and cancer-associated retinopathy

The deduced amino acid sequence of the recently cloned mouse 23kD photoreceptor cell-specific protein showed it to be identical to the recoverin protein and the CAR (cancer-associated retinopathy) protein. DNA sequence variants were found in the mouse recoverin gene (Rcvrn), and segregation analysis of restriction fragment length variants in recombinant inbred strains of mice assigned Rcvrn to mouse Chromosome (Chr) 11, between Sparc (3.7 map units) and Zfp-3 (2.3 map units). These results demonstrate a close linkage of recoverin to the tumor suppressor gene, Trp53. On the basis of these data, knowledge of the function of recoverin, and the characteristics of CAR, an experimentally testable model is presented to explain the molecular basis for CAR.

Localization of the murine activating transcription factor 4 gene to mouse chromosome 15.

Restriction fragment length variant analysis employing a mouse cDNA probe was used to localize the gene encoding murine activating transcription factor 4 (ATF-4) to mouse chromosome 15 in close proximity to Sis (the cellular homolog of the simian sarcoma virus oncoprotein). Previous studies suggest that conserved linkage relationships exist between this region of mouse chromosome 15 and human chromosome 22q. The chromosomal locations of genes encoding most members of the ATF and cyclic AMP response element binding protein (CREB) subfamily of b-zip proteins have not been determined. This study demonstrates that the location of the gene for murine ATF-4 is not linked to the genes for JUN family members, CREB1 and CREB2. Further mapping of individual ATF/CREB subfamily members in the mouse will provide insight into the evolution of this multigene family.

Genetic mapping of the integrin α1 gene ( Vla1) to mouse chromosome 13

The integrin alpha 1 chain (Vla1) associates with the beta 1 chain to form a heterodimer that functions as a dual laminin/collagen receptor in neural cells and hematopoietic cells. We have used an interspecies backcross gene-mapping technique to map the Vla1 gene to the distal end of chromosome 13 in the mouse genome. The Vla1 locus is located 3.5 cM distal to Ctla-3 and 7.8 cM distal to Htrla. We have further characterized this locus in recombinant inbred (RI) mice by examining the strain distribution patterns of nine genomic DNA restriction fragment length variants detected with alpha 1 cDNA probes. The RI gene mapping did not show linkage to previously mapped genes or mutants in the AXB, BXA, or AKXD RI sets and therefore defines a new genetic marker for the distal end of chromosome 13 in these RI sets.

A linkage map of mouse chromosome 19: Definition of comparative mapping relationships with human chromosomes 10 and 11 including the MEN1 locus

A linkage map of mouse Chromosome (Chr) 19 was constructed using an interspecific cross and markers defined by restriction fragment length variants. The map includes 20 markers, 9 of which had not been mapped previously in the mouse. The data further defined the relationship between genes on mouse Chr 19 and those on the long arm of human Chr 10 and the pericentric region of the long arm of human Chr 11. The comparative mapping analysis suggests that the proximal segment of mouse Chr 19 may contain the MEN1 locus and that the current study has identified additional genes that may be useful for positional cloning of this putative tumor suppressor gene.

Mapping of the mouse bilirubin UDP-glucuronosyltransferase gene (Gnt-1) to chromosome 1 by restriction fragment length variations.

Restriction endonuclease fragment length variations (RFLVs) were detected by using a rat cDNA probe for the bilirubin UDP-glucuronosyltransferase (UDPGT) gene between two mouse strains, 129/Sv and MOL-MIT. RFLVs of the gene were found by EcoRI and PvuII digestions. From linkage analyses of the three-point cross test using Elo and En-1 as marker genes, the bilirubin UDPGT gene was mapped at position 37 on chromosome 1. Bilirubin and phenol UDPGTs have been suggested to be expressed by a single gene by alternative splicing in human and rat. The mouse bilirubin UDPGT gene was named Gnt-1.

Restriction fragment length variants in the marsupial Sminthopsis crassicaudata.

A fully pedigreed colony of the dasyurid marsupial Sminthopsis crassicaudata has provided material for establishing two panels of DNA samples: a broadbased test panel and a two-generation family panel. These have been used to search for genetic markers in the form of restriction fragment length variants. The molecular probes--pSG-2H, a region of the S. crassicaudata embryonic beta-globin gene; pB8.BS, a region of the human ubiquitin gene, and p3-21a1:1, a region of the processed pseudogene of phosphoglycerate kinase-1 of the macropodid marsupial Macropus robustus--were hybridized to Southern blots of EcoR1-digested DNA from the panels. Analysis of these blots when probed with pSG-2H provided evidence of two alleles segregating at a single EcoR1 site. Analysis of the same blots when probed with pB8.BS suggested allelic variation at two closely linked EcoR1 sites. Probing the blots with p3-21a1:1 produced a complex pattern of bands resembling DNA fingerprints. The presence of a 12.3-kb band was found to conform to a simple autosomal dominant mode of inheritance. Analysis of the family data, for each probe, revealed no significant departure from Mendelian inheritance. This work has provided additional genetic markers that will enhance the use of S. crassicaudata as a model marsupial species and has demonstrated that a high level of genetic variability has been maintained in the marsupial colony.

A preliminary linkage map of the chicken genome

We have used backcross progeny from a cross between two inbred lines of chickens to construct a linkage map of the chicken. The map currently consists of 100 loci, identified using either anonymous cloned fragments of genomic DNA or sequences corresponding to cloned genes. Parent birds were derived from two lines of White Leghorn chickens, which differ in susceptibility to a number of diseases. Restriction fragment length variants were identified by comparison of the DNA of these two parent birds using a panel of seven restriction enzyme digests and the segregation pattern observed in progeny of these two birds. Restriction fragment length variants were detected for approximately 41% of the clones tested, whether these were known genes or random genomic fragments. This high level of variability was also reflected in the presence of variation within the parental lines for some clones. The overall size of the linkage groups and the progressively higher incidence of linkage as further clones were added suggests that the map covers the majority of the genome, although it is unlikely that there are marker loci on all the microchromosomes. The present map will be of use in locating genes affecting disease resistance, but also illustrates the relative ease with which such maps for the chicken can be constructed.

Identification of a putative γ-aminobutyric acid (GABA) receptor subunit rho 2 cDNA and colocalization of the genes encoding rho 2 (GABRR2) and rho 1 (GABRR1) to human chromosome 6q14–q21 and mouse chromosome 4

Screening of a genomic DNA library with a portion of the cDNA encoding the γ-aminobutyric acid (GABA) receptor subunit rho 1 identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho 1 gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho 1 gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to α, β, γ, and δ GABA receptor subunits and 74% similarity to the GABA rho 1 subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho 2. Polymerase chain reaction (PCR) amplification of rho 1 and rho 2 gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12–q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.

Genetic diversity and differentiation of indica and japonica rice detected by RFLP analysis

Genetic diversity and differentiation in indica and japonica groups of the cultivated rice (Oryza sativa L.) were studied by assaying DNA restriction fragment length polymorphisms of 12 indica and 14 japonica rice lines digested with three restriction endonucleases. A total of 49 probes were selected to represent the entire RFLP map at intervals of 20-30 cM. It was shown that 95 of the 145 possible probe/enzyme combinations, involving 43 probes and all three enzymes, detected restriction fragment length variation, and the degree of polymorphism varied greatly from one probe/enzyme combination to another. These results demonstrate that indica rice is genetically more diverse than japonica type. Significant differentiation between the two rice groups was detected by 33 probes representing 11 of the 12 rice chromosomes. It was deduced that the processes leading to differentiation involved a combination of molecular events that include base substitutions and insertion/deletions.

Localization of growth arrest-specific genes on mouse chromosomes 1, 7, 8, 11, 13, and 16.

Growth arrest in NIH3T3 cells is associated with increased expression of a variety of mRNAs, several of which have been isolated as cDNA clones. Six of these growth arrest-specific (Gas) genes were mapped by following the inheritance of DNA restriction fragment length variants (RFLVs) associated with them in panels of recombinant inbred (RI) strains of mice and in the progeny of backcrosses both between laboratory mouse strains and between a laboratory strain and Mus spretus. The six genes are unlinked. Gas-1 maps to Chromosome (Chr) 13, Gas-2 to Chr 7, Gas-3 to Chr 11, Gas-4 to Chr 16, Gas-6 to Chr 8, and Gas-10 to Chr 1.

Different Isozymes of Mouse 11/ß-Hydroxylase Produce Mineralocorticoids and Glucocorticoids

We have isolated and characterized two isozymes of mouse steroid 11 beta-hydroxylase (11 beta-OHase), designated 11 beta-OHase and aldosterone synthase (AS). Physical mapping of overlapping cosmid and phage isolates defined two genes (designated Cyp11b-1 and Cyp11b-2 in the standard nomenclature for cytochrome P450 genes) that are oriented in the same direction and separated by approximately 8 kilobase pairs of DNA. The two genes are highly homologous in their coding regions, with 84% nucleotide identity and 86% predicted amino acid identity. In regions where the sequences of the rat 11 beta-OHase and AS genes diverged most widely, the mouse sequences also differed significantly, thereby identifying putative mouse 11 beta-OHase and AS genes. Both genes were mapped to chromosome 15 by analyzing restriction fragment length variations in a panel of DNA samples from an interspecific cross. To determine the functional properties of the 11 beta-OHase and AS proteins, we transfected COS-7 cells with plasmids that expressed the proteins encoded by the 11 beta-OHase and AS genes. When expressed in transfected COS-7 cells, the 11 beta-OHase protein converted deoxycorticosterone to corticosterone but did not produce aldosterone. Consistent with its postulated role in mineralocorticoid biosynthesis, the product of the AS gene efficiently synthesized aldosterone. We next studied the expression of these two isozymes in Y1 adrenocortical tumor cells and in the intact mouse adrenal gland. Although Y1 cells otherwise resemble zona fasciculata cells and express the 11 beta-OHase gene at high levels, transcripts encoded by the AS gene were detected at levels approximately 10-fold lower than the 11 beta-OHase transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)

The mouse transition protein 1 gene contains a B1 repetitive element and is located on chromosome 1

The gene for mouse transition protein 1 (mTP1) was isolated, sequenced, and chromosomally mapped. The nucleotide sequence of 1895 bp of a 6.4-kb mTP1 genomic subclone was determined to include 788 bp of 5′ flanking region, 564 bp of coding region including a 396-bp intron and a TAA stop codon, and 543 bp of 3′ flanking region. The mTP1 gene contains a B1 repeat sequence within the only intron of the gene. The transcriptional start site of the mTP1 mRNA was determined to be located 31 bases upstream of the ATG translational start codon. Southern blot analysis demonstrated the presence of sequences homologous to the mTP1 cDNA in the genomes of the rat, hamster, bull, boar, dog, horse, ram, human, and two marsupials (the American opossum and Monodelphis), suggesting that the mTP1 gene sequence is widely conserved. The TP1 gene has been mapped by analysis of restriction fragment length variants (RFLV) in an interspecific backcross to a position 0.7 ± 0.4 cM telomeric of Mylf and 1.2 ± 0.5 cM centromeric of Vil on mouse chromosome 1.

Mapping genes encoding drug-metabolizing enzymes in recombinant inbred mice

Probes for cytochrome P450IVA (P450IVA), α- and π-class glutathione S-transferases (GST), and phenol-metabolizing UDP-glucuronyltransferase (UDPGT-K39) detected restriction fragment length variants (RFLVs) between C57BL 6J and DBA 2J mice. These variants were used to map the P450IVA genes ( Cyp4a) to chromosome 4, close to Mtv-13 and Pmv-19, midway between brown ( b) and Gpd-1; GSTα genes were mapped to chromosome 9, with a cross-hybridizing sequence mapping to another chromosome; the GSTπ genes were mapped to the distal end of chromosome 1 near Pmv-21; one UDPGT-K39 variant to chromosome 1, between Acrg and Emv-17, and another showed linkage to Odc-10 on an unidentified chromosome. No RFLVs were detected with probes for P450IID, P450 reductase, androsterone-metabolizing UDPGT, GSTμ, or microsomal GST.

Three brain sodium channel α-subunit genes are clustered on the proximal segment of mouse chromosome 2

We have used long-range physical mapping and restriction fragment length polymorphisms between two mouse species to determine the chromosomal organization and location of the genes encoding three distinct isoforms of the α-subunit of the brain sodium channel. Physical mapping by pulsed-field gel electrophoresis has established that Scn2a and Scn3a (genes encoding type II and type III sodium channel α-subunit isoforms) are physically linked and are separated by a maximum distance of 600 kb. The segregation of restriction fragment length variations in backcross progeny of a Mus musculus and Mus spretus mating indicates that Scn1a (gene encoding the type I sodium channel α subunit) and Scn2a are tightly linked and are separated by a distance of 0.7 cM. Linkage analysis in backcross and recombinant inbred (BXD and AKXD) strains of mice localized the three sodium channel genes to the proximal segment of mouse chromosome 2 and suggested the probable gene order centromere- Hc-Neb-Pmv7-Scn2a/Scn3a-Scn1a-Mpmv14. These results indicate that the three isoforms of the brain sodium channel α-subunit are encoded by three distinct genes that share a common ancestral origin.

Ras-like genes and gene families in the mouse

Four human RAS-like cDNAs and a mouse genomic DNA fragment were used to define novel mouse Ras-like genes and gene families. Inheritance of DNA restriction fragment length variants associated with these genes in recombinant inbred and backcross mice allowed definition of 12 genetic loci, nine of which were mapped, to chromosomes (Chr) 2, 4, 7, 8, 9, and 17. Two possible clusters of Ras-like and/or G protein genes were identified, on Chrs 9 and 17.

A linkage map of mouse chromosome 8: Further definition of homologous linkage relationships between mouse chromosome 8 and human chromosomes 8, 16, and 19

Using an interspecific cross, a mouse chromosome 8 linkage map spanning 72 cM has been defined by the segregation of restriction fragment length variants. Linkage and genetic distance were established for 10 loci by analysis of 114 meiotic events and indicated the following gene order: (centromere)- Insr-3.5 cM- Plat-26.3 cM- Crryps/Mel/Jund-3.5 cM- Junb/Ucp-10.5 cM- Mt-1-27.2 cM- Acta2-0.9 cM- Aprt. These data provide further definition of mouse chromosome 8 linkage relationships and the relationship between segments of this chromosome and human chromosomes 8, 16, and 19.

Analysis of second-step mutations of class II and class III CHOaprt heterozygotes: Chromosomal differences in deletion frequencies

We have determined the nucleotide sequence surrounding a BclI restriction fragment length variation near the aprt gene of CHO cells. By BclI digestion of the PCR-amplified DNA from a variety of APRT-deficient mutants derived from CHO cells, we were able to infer the following. First, all three heterozygotes of class II, which are known to undergo the second mutational step via a large deletion event occurring at high frequency, are mutant at the chromosome Z4-linked allele, and wild type at the Z7 allele. Second, both class-III heterozygotes, which mutate to the APRT- phenotype at low frequency, are mutant at the Z7 allele, wild type at the Z4 allele. A total of 12 class-I lines, defined as having already undergone a deletion event and yielding fully APRT- mutants at low frequency were all found to have lost the Z7-linked allele. We conclude that the Z7-linked allele is substantially more susceptible to mutation by the large deletion event than is the Z4-linked allele. This supports a hypothesis we advanced earlier to explain the existence of the class-III heterozygotes but does not support previous work suggesting that a chromosomal inversion break-point junction near the Z4-linked aprt allele is responsible for the high frequency deletion event.

A proximal mouse chromosome 9 linkage map that further defines linkage groups homologous with segments of human chromosomes 11, 15, and 19

A 42-cM map of proximal mouse chromosome 9, including eight loci defined by restriction fragment length variants, has been generated. Linkage was established by haplotype analyses of 114 interspecific backcross mice and indieated the following gene order: (centromere) Pvs-5.3 cM-Icam-1 Ldlr-18.4 cM- Thy-1-1.8 cM- Ncam-0.9 cM- Hexa-7.9 cM- Gsta-7.9 cM- Trf. Three of these loci, Pvs, Icam-1, and Hexa, have not been mapped previously. Together with previous mapping studies the current results suggested that chromosomal segments of mouse chromosomes 7 and 9 and chromosomal segments of human chromosomes 11, 15, and 19 derive from a single putative primordial chromosome. The studies support the postulate that detailed analysis of chromosome organization will be useful in defining events in mammalian evolution.

Assignment of the perlecan (heparan sulfate proteoglycan) gene to mouse Chromosome 4

Basement membranes are sheets of extracellular matrix produced by epithelial and endothelial cells which separate these cells from underlying connective tissue. The primary constituents of basement membranes are collagen type IV, laminin and entactin (reviewed by Timpl 1987) and perlecan, a heparan sulfate proteoglycan (Ledbetter et al. 1985). These components interact with each other to maintain the structural integrity of the matrix. Perlecan (Plc) has been shown to modify cell attachment (Clement et al. 1989) and is likely to be involved in the ionic filtration performed by the glomerular basement membrane (Kanwar et al. 1980). The core protein of perlecan has an approximate size of Mr = 400,000 (Ledbetter et al. 1985). Recently, the mouse perlecan cDNA has been partially cloned and sequenced (Noonan et al. 1989). The perlecan gene has been localized to human chromosome (chr) lp36. l by in situ hybridization using the mouse perlecan cDNA probe BPG-7 [a 2193 base pair (bp) mouse perlecan cDNA insert] (Wintle et al. 1990). In this report we demonstrate the presence of DNA sequence variants in the mouse perlecan (Plc) gene. In addition, we localized perlecan to mouse Chr 4 by segregation analysis of restr ic t ion fragment length variants (RFLVs) in recombinant inbred (RI) strains of mice. EcoR I and B a m H I restriction endonuclease digests of DNA from five progenitor mice strains (AKR/ J, DBA/2J, C57L/J, C57BL/6J and C3H/HeJ) were tested for RFLVs in the Ptc gene by Southern blot hybridization using the BPG-7 probe (Fig. 1). Two alleles were evident. In the DNA digests of AKR/J, DBA/2J and C3H/HeJ strains, two DNA fragments, approximately 7.4 kb (faint band) and 6.4 kb (intense band) in size, were detected. The alternative pattern seen in C57L/J and C57BL/6J consisted of 5.8 kb (intense band), 10.4 kb and 1.9 kb (faint bands) DNA fragments. A 2.2 kb DNA fragment was common to all