Linkage of isozyme loci in the mouse: Phosphoglucomutase-2 (Pgm-2), mitochondrial NADP malate dehydrogenase (Mod-2), and dipeptidase-1 (Dip-1)

The linkages of the isozyme genes Mod-2, Pgm-2, and Dip-1 have been determined in tests with established linkage group markers among inbred strains of mice. Unique alleles for both Mod-2 and Pgm-2 have been observed in the strain of SM/J. Linkage was determined from backcross progeny of the matings C57BL/6J×(SM/J×C57BL/6J)F₁, (SM/J×SWR/J)F₁×SM/J, and (SM/J×SWR/J)F₁×SJL/J. The gene Mod-2 is on linkage group 1. In a three-point cross of the loci Gpi-1, c, and Mod-2, the c locus was determined to be the middle gene. No double crossovers were observed. Our combined data show the following linkages: Gpi-1 to c, 28.3±3.2%; Gpi-1 to Mod-2, 33.3±3.0%; and c to Mod-2, 4.1±2.8%. The proposed gene order for four markers on LG I is Gpi-1-p-c-Mod-2. The gene Pgm-2 was linked to Gpd-1 (27.0±4.2%) on LGVIII. Two backcrosses segregating for Pgm-2 and b, (SM/J×DBA/2J) F₁×DBA/2J and (SM/J×DBA/2J)F₁×C57BR/cdJ, showed 9.1±4.3% recombination. The proposed gene order on LG VIII is b-Pgm-2-Gpd-1. The genes Pgm-1 and Pgm-2 are not linked (53.4±4.4%). Linkage of the isozyme genes Dip-1 and Id-1 on LG XIII was observed in backcross progeny of the crosses (SJL/J×C57BL/6J)F₁×SJL/J and C57BL/6J×(SM/J×C57BL/6J)F₁. The combined recombination was 23.8±2.8%. Two cases are established where genes whose enzyme products share substrate affinities (Pgm-1 and Pgm-2; Mod-1 and Mod-2) are not linked. Our data generally support the conclusion that functionally or metabolically related isozyme genes are not contiguous on mouse linkage groups.This investigation was supported in part by Public Health Service General Research Support Grant GM-09966 and in part by Public Health Service Training Grant 5T01 HD-00032-07 from the National Institute of Child Health and Human Development, and by Atomic Energy Commission contract AT(30-1)-3671.     

Mapping in the mouse of the region homologous to the rat growth and reproduction complex (grc)

Offprint requests: J. Klein.     

Sequences homologous to the rat ID element in mouse DNA

Sequences homologous to the rat frain specific identifier sequences were found in the mouse genome. These sequences were defined by dot- and blotting-hybridisation in different DNA and RNA preparations. It was shown that there are (1-2) X 10(3) copies of these sequences per genome. They are not transcribed in mouse brain and possibly are not connected with mouse brain specific genes expression.     

Assignment of 12 loci to rat chromosome 5: evidence that this chromosome is homologous to mouse chromosome 4 and to human chromosomes 9 and 1 (1p arm)

Twelve loci have been assigned to rat chromosome 5: aldolase B (ALDOB), atrial natriuretic factor (ANF = pronatriodilatin, PND), D4RP1, DSI1, galactosyltransferase (GGTB2), glucose transporter (GLUT1), interferon alpha 1 and related interferon alpha (INFA), interferon beta (INFB), lymphocyte-specific protein-tyrosine kinase (LCK), oncogene MOS, alpha 2U-globulin (major urinary protein, MUP), and orosomucoid (ORM, also called alpha 1-acid glycoprotein, AGP). Among these, the interferon alpha and beta genes map in the q22-23 region, which also contains a transformation suppressor gene (SAI1). The other loci reside outside this region. This study also indicated that the rat genome contains 2 LCK genes, unlike the human and murine genomes. These new assignments on rat chromosome 5 demonstrate that this chromosome is highly homologous to mouse chromosome 4 and carries synteny groups conserved on human chromosome 9 (interferon alpha and beta, galactosyltransferase, orosomucoid, and aldolase B genes) and on the short arm of human chromosome 1 (MYCL, glucose transporter, protein kinase LCK, and atrial natriuretic factor genes).     

Polymorphic class II sequences linked to the rat major histocompatibility complex (RT1) homologous to human DR and DQ sequences

Until recently, the analysis of Class II genes linked to the rat major histocompatibility complex, RT1, has been confined to serologic and electrophoretic analysis of their gene products. To obtain a more definitive estimate of the number and relative polymorphism of RT1 Class II sequences, we performed Southern blot analysis of rat genomic DNA employing human cDNA probes specific for Class II heavy and light chain genes. Southern blots of EcoRI and BamHI digests of genomic DNA from ten inbred strains, expressing eight RT1 haplotypes, were hybridized with the human DQ beta or DR beta cDNA that are homologous to Class II light chain sequences. Four to eight bands were observed to hybridize with the light chain cDNA: band sizes ranged from 2.5 to 28 kb. Restriction fragment patterns were polymorphic; the only identical patterns observed were those associated with RT1 haplotypes with identical RT1.B regions. The number and size of bands hybridizing with DQ beta and DR beta suggested a minimum of four light chain sequences in each haplotype. Southern blots of BamHI and EcoRI digests of genomic DNA from the same strains were hybridized with a DR alpha cDNA that is homologous to Class II heavy chain sequences. All RT1 haplotypes expressed either a 10.0-kb or 13.0-kb band when digested with BamHI, and either a 17-kb or 3.7-kb band when digested with EcoRI. Considerably less polymorphism was detected with the DR alpha probe; this observation is consistent with previously reported limited protein polymorphism of the rat equivalent of the I-E alpha subunit. The size and number of bands hybridizing with the DR alpha probe suggests a minimum of two heavy chain sequences. These observations suggest that the RT1 complex includes more Class II sequences than have been observed in serologic and electrophoretic analyses of Class II gene products. Furthermore, the level of polymorphism of RT1 Class II sequences appears to be comparable with mouse and human Class II sequences.     

Sequences homologous to the human D1S1 locus present on human chromosome 3.

We have investigated the segregation, in somatic cell hybrids, of the human D1S1 locus, previously assigned to 1p36 by in situ hybridization. We have shown that the clone which defines this locus, lambda Ch4A-H3, originates from human chromosome 3, but contains a 1.7-kilobase (kb) PstI-HindIII repetitive element that is also present on chromosome 1, probably distal to PGD. The clone recognizes restriction fragment length polymorphisms within the single-copy sequence on chromosome 3 and one for the enzyme StuI in the repeated sequence on chromosome 1. These experiments thus expose a level of complexity in the D1S1 locus not revealed by earlier in situ hybridization studies.     

Expression of PCSK1 (PC1/3), PCSK2 (PC2) and PCSK3 (furin) in mouse small intestine

The family of serine proteases known as the proprotein convertases subtilisin/kexin type (PCSK) is responsible for the cleavage and maturation of many precursor hormones. Over its three successive regions, the duodenum, the jejunum and the ileum, the small intestine (SI) expresses over 40 peptide hormones necessary for normal intestinal physiology. Most of these hormones derive from proteolytic cleavage of their cognate inactive polypeptide precursors. Members of the PCSK family of proteases have been implicated in this process, although details of enzyme-substrate interactions are largely lacking. As a first step towards elucidating these interactions, we have analyzed by immunohistochemistry the regional distribution of PCSK1, PCSK2 and PCSK3 in mouse SI as well as their cellular co-localization with substance P (SP), cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide (GIP) and somatostatin (SS), 4 peptide hormones known to result from PCSK-mediated processing. Results indicate that PCSK1 is found in all three regions of the SI while PCSK2 and PCSK3 are primarily expressed in the upper two, the duodenum and the jejunum. In these proximal regions, PCSK1 was detectable in 100% of SP-positive (+) cells, 85% of CCK+ cells and 50% of GIP+ cells; PCSK2 was detectable in 40% of SS+ cells and 35% of SP+ cells; PCSK3 was detectable in 75% of GIP+ cells and 60% of SP+ cells. These histological data suggest that the 3 PCSKs may play differential and overlapping roles in prohormone processing in the three regions of the SI.     

Assignment of the human renal dipeptidase gene (DPEP1) to band q24 of chromosome 16.

Renal dipeptidase (DPEP1) or dehydropeptidase-I (E.C.3.4.13.11) is a kidney membrane enzyme which hydrolyzes a variety of dipeptides. DPEP1 is implicated in the renal metabolism of glutathione and its conjugates and is also responsible for hydrolysis of beta-lactam antibiotics. Using a human DPEP1 cDNA probe, we mapped the human renal dipeptidase gene to human chromosome 16 at band q24 by in situ hybridization.     

Physicochemical and biological properties of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose (6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin)

A unique multibranched cyclomaltooligosaccharide (cyclodextrin, CD) of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose [6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin, (G(2))(3)-betaCD] was prepared. The physicochemical and biological properties of (G(2))(3)-betaCD were determined together with those of monobranched CDs (6-O-alpha-D-glucopyranosyl-alpha-cyclodextrin (G(1)-alphaCD), 6-O-alpha-D-glucopyranosyl-beta-cyclodextrin (G(1)-betaCD), and 6-O-alpha-maltosyl-beta-cyclodextrin (G(2)-betaCD)). NMR spectra of (G(2))(3)-betaCD were measured using various 2D NMR techniques. The solubility of (G(2))(3)-betaCD in water and MeOH-water solutions was extremely high in comparison with nonbranched betaCD and was about the same as that of the other monobranched betaCDs. The formation of an inclusion complex of (G(2))(3)-betaCD with stereoisomers (estradiol, retinoic acid, quinine, citral, and glycyrrhetinic acid) depends on the cis-trans isomers of guest compounds. The cis isomers of estradiol, retinoic acid, and glycyrrhetinic acid were included more than their trans isomers, while the trans isomers of citral and quinine fit more tightly than their cis isomers. (G(2))(3)-betaCD was the most effective host compound in the cis-trans resolution of glycyrrhetinic acid. Among the branched betaCDs, (G(2))(3)-betaCD exhibited the weakest hemolytic activity in human erythrocytes and showed negligible cytotoxicity in Caco-2 cells up to 200 microM. These results indicate unique characteristics of (G(2))(3)-betaCD in some biological responses of cultured cells.     

Molecular and functional analyses of the human and mouse genes encoding AFG3L1, a mitochondrial metalloprotease homologous to the human spastic paraplegia protein

The identification of SPG7 as the gene defective in a recessive form of spastic paraplegia has drawn attention to the yeast protein family of ATP-dependent zinc metalloproteases. The protein encoded by SPG7, paraplegin, shows high homology to members of this protein family. Recently, many mammalian ATP-dependent zinc metalloproteases have been identified and considered as possible candidates for defects in other forms of hereditary spastic paraplegia and possibly other neurodegenerative disorders. So far only a partial sequence has been available for one of those genes, ATPase family gene-3, yeast-like-1 (AFG3L1). We have carried out detailed molecular analysis of this gene and identified and characterized its mouse orthologue, Afg3l1. Our data indicate that AFG3L1 is transcribed into four mRNA isoforms that are not translated in humans. Afg3l1 encodes a protein with high homology to paraplegin and the other members of the ATP-dependent zinc metalloprotease family. Like the other ATP-dependent zinc metalloproteases, Afg3l1 localizes to the mitochondria.     

The purification and properties of human alpha1-antitrypsin (alpha1-antiprotease), variant Z.

After three stages of preliminary purification, variant Z was chromatographed on a DEAE-cellulose column. Upon elution with a linearly increasing concentration of NaCl, variant Z was recovered in two separate peaks, the first of which contained 81% and the second 19% of the total. The preparation corresponding to the first peak was homogeneous by various criteria. The trypsin and chymotrypsin inhibiting capacities and the specific antigenic activity of the preparation were nearly the same as those of an authentic sample of variant M. Variant Z contained 8 or 9 more gycine residues than variant M, but no appreciable difference was found between their carbohydrate contents. By analytical isoelectrofocusing the isoinhibitors of purified variant Z overlapped with those in the plasma of the donor and were cathodal to, but partially overlapped with purified variant M. After desialysation, the overlap between the different variants became complete, but variant Z contained a larger proportion of cathodal and smaller proportion of anodal components than variant M. Both variants formed five distinct isoinhibitor-protease complexes after incubation with trypsin and chymotrypsin and the corresponding complexes in the different variants completely coincided.     

Assignment of three rat integrin genes to Chromosome 19 (ITGB1), Chromosome 3 (ITGA4), and Chromosome 7 (ITGA5)

By means of somatic cell, hybrids segregating rat chromosomes, we determined the chromosome localization of three rat 1 family integrin genes. ITGB1 was assigned to Chromosome (Chr) 19, ITGA4 to Chr 3, and ITGA5 to Chr 7. These chromosome assignments reveal or confirm homology between two pairs of rat and human chromosomes (rat Chr 3-human Chr 2; rat Chr 7-human Chr 12).     

A human genomic sequence highly homologous to the 3'-untranslated region of rat uricase mRNA

A human genomic sequence was isolated from a library using a rat uricase cDNA probe. Sequence analysis has shown that it is highly homologous to the 3'-untranslated region of rat uricase mRNA. Total loss of uricase activity in human is, therefore, not due to total loss of the gene. Discovery of high degree of conservation of the non-coding region of the gene would be of great interest as we attempt to learn the process of gene evolution.