Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The K _m values for ethanol and NAD⁺, approximately 0.4 and 0.03mm, respectively, of enzyme purified from both strains are similar. Moreover, the K _i for NADH, 1 µm, the pH optimum for ethanol oxidation, 10.5, and the V _max for ethanol oxidation, 160 min^–1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.Supported by NIAAA Grant AA 04307.     

Analysis of the mouseAmy locus in recombinant inbred mouse strains

Pancreatic and salivary amylase cDNA probes have been used to search for new DNA fragment length variation among a total of 43 inbred mouse strains. Fragment length differences found with three restriction endonucleases grouped the strains into two major classes. The segregation of these variant fragments has been analyzed among several sets of recombinant inbred strains and is presented here. Previously reported differences for strains YBR and CE have been confirmed. New segregation data for carbonic anhydrase, a locus near the proximal end of mouse chromosome 3, are presented.     

Genetic analysis of nonhistone chromosomal protein inheritance in recombinant inbred mouse strains using two-dimensional electrophoresis

Analysis of hepatic nonhistone chromosomal protein (NHCP) expression in male mice from progenitor strains (C3H/HeN, C57BL/6N), their F₁ hybrid (B6C3), and seven recombinant inbred strains (RIs) (B6N×C3N) by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) detected 16 NHCPs whose expression in RIs could be correlated to each other and with strain distribution patterns (SDP) of 20 genetic markers differing in the progenitors. Of the 400₊ NHCP spots detected in RI 2D-PAGE maps, 172 were common to progenitors and all RIs. There was a characteristic absence of five NHCPs in one RI, Y. Ten C3H-specific and six C57-specific NHCP inherited in B6C3 also appeared in RIs. The SDP of C3H-specific NHCP 2 matched the SDP of beta-glucuronidase on chromosome 5 and carbonic anhydrase on chromosome 3, and C57-specific NHCP 5 SDP corresponded to that for nonagouti trait on chromosome 2. These 16 NHCP genetic marker inheritance differences detected in RIs add to the 23 previously established genetic marker differences between the progenitors.This study was supported in part by funds from NIH Grants CA 33305 and CA 16672 and Exxon Corporation, USA.     

Gene markers for alcohol-metabolizing enzymes among recombinant inbred strains of mice with differential behavioural responses towards alcohol

The genetic variability of alcohol dehydrogenase (C₂ isozyme), aldehyde dehydrogenase (A₂ isozyme) and aldehyde oxidase (A₂ isozyme) has been examined among recombinant inbred strains of mice which have been previously studied concerning their differential behavioural responses towards alcohol. The results showed no correlation between biochemical phenotype for these loci and behavioural response.     

Frequency of X-Y chromosome dissociation in mouse spermatocytes from interstrain crosses,recombinant inbred strains,and chimeras: Possible involvement of paternal genome imprinting

The frequency of dissociation of the X-Y chromosome bivalent in diakinesis-metaphase I spermatocytes differs significantly between two inbred mouse strains, CBA (29%) and KE (7%), that were used to obtain reciprocal F1 hybrids, and to develop recombinant inbred (R1) strains. The level of X-Y dissociation was significantly higher in (KExCBA)F1 hybrids sired by the CBA males (24%) than in reciprocal F1 hybrids (12%), revealing the inheritance after the father. Among 14 RI strains, nine were concordant with KE, one with CBA, and four had intermediate phenotype, significantly different from both progenitor strains. This shows that at least two genes are involved, and their possible linkage with agouti and Trf loci is suggested. The linkage with agouti was confirmed by testing additional 10 CBXE incipient RI strains. There was no significant difference in the level of X-Y dissociation between EXCB RI strains derived from the original cross sired by the CBA males and CBXE RI strains derived from the reciprocal cross. The involvement of the Y chromosome-linked factors was unlikely because it was found earlier (Krzanowska, 1989: Gamete Res 23:357–365) that two congenic strains, KE and KE.CBA, differing with respect to the source of the Y chromosome, had the same level of X-Y dissociation. Thus, the difference obtained between reciprocal F1 hybrids is interpreted in terms of paternal genome imprinting imposed by CBA males and propagated only in the presence of some alleles derived from this strain. Analysis of six KE ? CBA-T6 chimeras, among them three germ line chimeras, points to the conclusion that the tendency to low or high level of X-Y chromosome dissociation is expressed rather autonomously by KE or CBA-T6 spermatocytes (as recognized by a marker chromosome pair), respectively, and was not modified by the presence of somatic cells of the opposite strain. © 1994 Wiley-Liss, Inc.     

Enzyme markers in inbred rat strains: Genetics of new markers and strain profiles

Twenty-six inbred strains of the laboratory rat (Rattus norvegicus) were examined for electrophoretic variation at an estimated 97 genetic loci. In addition to previously documented markers, variation was observed for the enzymes aconitase, aldehyde dehydrogenase, and alkaline phosphatase. The genetic basis of these markers (Acon-1, Ahd-2, and Akp-1) was confirmed. Linkage analysis between 35 pairwise comparisons revealed that the markers Fh-1 and Pep-3 are linked. The strain profiles of the 25 inbred strains at 11 electrophoretic markers are given.     

Difference in anxiety and sensitization of the acoustic startle response between the two inbred mouse strains BALB/cAN and DBA/2N

The inbred mouse strain BALB has been proposed to be an animal model for pathological anxiety. BALB exhibits a stronger acoustic startle response (ASR) than the 'less emotional' inbred strain DBA. Four experiments were conducted to determine whether this strong ASR is due to a higher anxiety level and/or to greater sensitization in BALB than in DBA, with the following results: (1) The ASR to the very first startle stimulus was found to be much stronger in BALB than in DBA, and freezing behavior evoked by startle stimuli was more pronounced in BALB than in DBA. These findings indicate a higher level of anxiety in this strain. (2) ASR amplitudes of BALB initially rose much higher during consecutive startle stimuli and remained at a high level much longer than in DBA. Thereafter, ASR amplitude dropped more slowly and to a lesser degree than in DBA. Startle amplitudes decreased similarly in both strains (strong exponential decrease) only when a low sound pressure level (SPL) was used which elicited approximately the same low ASR in both strains. These results can only be explained by increased sensitization in BALB. (3) The slope of the i/o-function, which represents the relation between sensory input and motor output, was steeper in BALB than in DBA. As it has been shown recently, sensitization increases the slope of the startle i/o-function indicating increased sensitization in BALB. It is discussed, however, whether anxiety also contributes to this effect. (4) Footshocks increased the ASR much less in BALB than in DBA, again showing increased sensitization in BALB. Both a higher level of anxiety and greater sensitization therefore determined the greater strength of the ASR in BALB than in DBA.     

The relationship between nephron number,kidney size and body weight in two inbred mouse strains

While some reports in humans have shown that nephron number is positively correlated with height, body weight or kidney weight, other studies have not reproduced these findings. To understand the impact of genetic and environmental variation on these relationships, we examined whether nephron number correlates with body weight, kidney planar surface area, or kidney weight in two inbred mouse strains with contrasting kidney sizes but no overt renal pathology: C3H/HeJ and C57BL/6J. C3H/HeJ mice had smaller kidneys at birth and larger kidneys by adulthood, however there was no significant difference in nephron number between the two strains. We did observe a correlation between kidney size and body weight at birth and at adulthood for both strains. However, there was no relationship between nephron number and body weight or between nephron number and kidney size. From other studies, it appears that a greater than 2-fold variation is required in each of these parameters in order to demonstrate these relationships, suggesting they are highly dependent on scale. Our results are therefore not surprising since there was a less than 2-fold variation in each of the parameters examined. In summary, the relationship between nephron number and body or kidney size is most likely to be demonstrated when there is greater phenotypic variation either from genetic and/or environmental factors.     

The relationship between nephron number, kidney size and body weight in two inbred mouse strains

While some reports in humans have shown that nephron number is positively correlated with height, body weight or kidney weight, other studies have not reproduced these findings. To understand the impact of genetic and environmental variation on these relationships, we examined whether nephron number correlates with body weight, kidney planar surface area, or kidney weight in two inbred mouse strains with contrasting kidney sizes but no overt renal pathology: C3H/HeJ and C57BL/6J. C3H/HeJ mice had smaller kidneys at birth and larger kidneys by adulthood, however there was no significant difference in nephron number between the two strains. We did observe a correlation between kidney size and body weight at birth and at adulthood for both strains. However, there was no relationship between nephron number and body weight or between nephron number and kidney size. From other studies, it appears that a greater than two-fold variation is required in each of these parameters in order to demonstrate these relationships, suggesting they are highly dependent on scale. Our results are therefore not surprising since there was a less than two-fold variation in each of the parameters examined. In summary, the relationship between nephron number and body or kidney size is most likely to be demonstrated when there is greater phenotypic variation either from genetic and/or environmental factors.     

Comparison of strains and culture media used for mouse in vitro fertilization

Success rates of superovulation in response to gonadotropic hormone treatment and in vitro fertilization (ie, mitotic cleavage following insemination) of mouse eggs from outbred CD-1, hybrid CB6F_l, or hybrid B6CBAF₁, mice were compared using either a mouse inseminationmedium, modified Krebs-Ringer-bicarbonate (m-KRB), or a human insemination medium, Ham's F10 nutrient mixture. Inseminations were performed in either organ culture dishes or screw-top, flat-side tissue culture tubes. Mean superovulation rates (± SD) were 24.2 (5.1) for CD-1, 33.0 (5.8) for CB6F₁, and 16.3 (6.6) for B6CBAF₁ mice. For in vitro cleavage the best combination of mouse strain, insemination medium, and culture container was achieved using CB6F₁, mice, m-KRB medium, and culture tubes. However, Ham's medium used with either hybrid mouse strain was shown to be employable for fertilization of mouse eggs in vitro as a quality control assay and/or experimental model system for testing the human in vitro fertilization procedure.     

Genetic map of AFLP markers in the rat (Rattus norvegicus) derived from the H x B/Ipcv and B x H/Cub sets of recombinant inbred strains

The amplified fragment length polymorphism (AFLP) technique has been used to enhance marker density in a large set of recombinant inbred strains (H × B and B × H) derived from a spontaneously hypertensive rat (SHR/OlaIpcv) and a Brown-Norway (BN.lx/Cub) inbred strain. Thirteen different primer combinations were tested and a total of 191 polymorphic bands were detected. From these polymorphic bands 89 AFLP markers could be assigned to specific chromosomes. Several of these AFLP markers were mapped to regions with low marker density, thus filling up gaps in the existing genetic map of these recombinant inbred strains. These results substantiate the value of the AFLP technology in increasing marker density in genetic maps.     

Linkage of genes for laminin B1 and B2 subunits on chromosome 1 in mouse

Summary We have used cDNA clones for the B1 and B2 subunits of laminin to find restriction fragment length DNA polymorphisms for the genes encoding these polypeptides in the mouse. Three alleles were found forLamB2 and two forLamB1 among the inbred mouse strains. The segregation of these polymorphisms among recombinant inbred strains showed that these genes are tightly linked in the central region of mouse Chromosome 1 betweenSas-1 andLy-m22, 7.4±3.2 cM distal to thePep-3 locus. There is no evidence in the mouse for pseudogenes for these proteins. This work was supported by U. S. Public Health Service Grant GM28464 to R.W.E. Editor's Statement Investigation into the biosynthesis of laminin indicates that several different polypeptides are assembled to form the intact molecule. This paper represents an extension of previous work which takes a genetic approach to further define the relationships among the polypeptides involved. Gordon H. Sato     

Leishmania tropica: pathogenicity and in vitro macrophage function in strains of inbred mice.

Of seven strains of inbred mice and one hybrid that were infected intracutaneously with 5, 10, or 20 × 10⁶ active promastigotes of Leishmania tropica major, two strains (CBA/Ca and C₃H/He) recovered from the infection and their lesions healed within 3 to 5 months. The other strains, with the possible exception of C57B1/6 animals, remained infected, carrying large cutaneous ulcers throughout their lives. These included DBA/2, A/Jax, Balb/c, athymic nude mice of Balb/c origin (nu/nu) and the heterozygote Balb/c (nu⁺). The responses of C57B1/6 animals were of intermediate type with a tendency toward nonhealing at higher doses of the parasite. The cutaneous infection of athymic nude mice invariably gave rise to fulminating visceral infections and death. This condition was never observed in the other strains tested. Concanavalin A (Con A)-stimulated syngeneic or allogeneic lymphocytes of intact mice activated peritoneal macrophages of both healer and nonhealer mice, resulting in complete destruction of phagocytosed L. enriettii within 24 to 48 hr. The destruction of ingested L. tropica was confined to macrophages of healer mice and required 72 to 96 hr to reach completion. However, removal of Con A-stimulated lymphocytes from macrophage cultures and regular pulsing of the cells with a lymphokine-rich supernatant produced a state of sustained activation, resulting in destruction of L. tropica inside macrophages of both healer and nonhealer mice. The ability of Con A-stimulated lymphocytes of nonhealer animals to induce effective levels of activation in healer macrophages on one hand, and eventual destruction of L. tropica in macrophages of nonhealer mice under condition of sustained activation on the other, had indicated that so far as the in vitro situation is concerned, there is no inherent defect in lymphocytes or macrophages of nonhealer animals, although the threshold of activation necessary for killing of the parasite seems to be higher for cells of nonhealer origin.     

Histidine decarboxylase phenotypes of inbred mouse strains: A regulatory locus (Hdc) determines kidney enzyme concentration

Mouse kidney histidine decarboxylase (HDC) provides a model system to study genetic control of a hormone-regulated enzyme (inducible by estrogen and thyroxine; repressible by testosterone). Five major HDC phenotypes scored on the basis of (i) enzyme activity and (ii) the difference in activity between the sexes (females usually higher than males) have been discovered by screening 38 strains of mice. One genetic difference between high-activity strains (DBA/2 and C3H/He) and low-activity strains (C57BL/6 and C57BL/10) has been examined in detail. The phenotypic difference segregates as a single gene in both conventional crosses and between recombinant inbred (RI) strains. Immunoprecipitation has shown that the activity difference is due to an alteration in the number of enzyme molecules. The phenotypic difference between high and low strains can therefore be attributed to different alleles of a single regulatory locus, Hdc; the alleleHdc ^d determines low HDC concentration, and the allele Hdc ^d high concentration. Hdc has been mapped to chromosome 2 using data from both comparisons of strain distribution patterns of previously mapped loci within RI strains and a conventional three-point cross. The probable gene order is B2m-pa-Hdc, with map distances of 3.1±1.7 and 2.0±1.4 cM, respectively.This work was supported by an MRC project grant to Grahame Bulfield, an SERC research studentship to S. A. M. Martin, and NIH Research Grant GM 18684 from the National Institute of General Medical Sciences to B. A. Taylor. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory animal care.     

Trypanosoma cruzi: In vivo and in vitro correlation between T-cell activation and susceptibility in inbred strains of mice

The in vivo susceptibility of several inbred strains of mice to the Y and CL strains of Trypanosoma cruzi was compared to the in vitro ability of spleen cells from infected mice to generate factor(s) able to activate macrophages to a trypanocidal state. Spleen cells from resistant immune mice generate higher levels of the factor(s) and do so at earlier times during infection than those of susceptible mice. The spleen cells capable of generating the in vitro factor(s) are also capable of conferring resistance upon passive transfer. Removal of immunoglobulin-bearing cells from the immune spleen cell population did not affect either transfer of protection in vivo or generation of the factor(s) in vitro. The cellular basis underlying the differences between susceptible and resistant mouse strains has not yet been determined.     

Identification of genetic determinants of IGF‐1 levels and longevity among mouse inbred strains

The IGF‐1 signaling pathway plays an important role in regulating longevity. To identify the genetic loci and genes that regulate plasma IGF‐1 levels, we intercrossed MRL/MpJ and SM/J, inbred mouse strains that differ in IGF‐1 levels. Quantitative trait loci (QTL) analysis of IGF‐1 levels of these F2 mice detected four QTL on chromosomes (Chrs) 9 (48 Mb), 10 (86 Mb), 15 (18 Mb), and 17 (85 Mb). Haplotype association mapping of IGF‐1 levels in 28 domesticated inbred strains identified three suggestive loci in females on Chrs 2 (13 Mb), 10 (88 Mb), and 17 (28 Mb) and in four males on Chrs 1 (159 Mb), 3 (52 and 58 Mb), and 16 (74 Mb). Except for the QTL on Chr 9 and 16, all loci co‐localized with IGF‐1 QTL previously identified in other mouse crosses. The most significant locus was the QTL on Chr 10, which contains the Igf1 gene and which had a LOD score of 31.8. Haplotype analysis among 28 domesticated inbred strains revealed a major QTL on Chr 10 overlapping with the QTL identified in the F2 mice. This locus showed three major haplotypes; strains with haplotype 1 had significantly lower plasma IGF‐1 and extended longevity (P < 0.05) than strains with haplotype 2 or 3. Bioinformatic analysis, combined with sequencing and expression studies, showed that Igf1 is the most likely QTL gene, but that other genes may also play a role in this strong QTL.     

The mouse antibody heavy chain repertoire is germline-focused and highly variable between inbred strains

The human and mouse antibody repertoires are formed by identical processes, but like all small animals, mice only have sufficient lymphocytes to express a small part of the potential antibody repertoire. In this study, we determined how the heavy chain repertoires of two mouse strains are generated. Analysis of IgM- and IgG-associated VDJ rearrangements generated by high-throughput sequencing confirmed the presence of 99 functional immunoglobulin heavy chain variable (IGHV) genes in the C57BL/6 genome, and inferred the presence of 164 IGHV genes in the BALB/c genome. Remarkably, only five IGHV sequences were common to both strains. Compared with humans, little N nucleotide addition was seen in the junctions of mouse VDJ genes. Germline human IgG-associated IGHV genes are rare, but many murine IgG-associated IGHV genes were unmutated. Together these results suggest that the expressed mouse repertoire is more germline-focused than the human repertoire. The apparently divergent germline repertoires of the mouse strains are discussed with reference to reports that inbred mouse strains carry blocks of genes derived from each of the three subspecies of the house mouse. We hypothesize that the germline genes of BALB/c and C57BL/6 mice may originally have evolved to generate distinct germline-focused antibody repertoires in the different mouse subspecies.