Increased Ap<Subscript>4</Subscript>A levels and ecto-nucleotidase activity in glaucomatous mice retina

The pathogenesis of glaucoma involves numerous intracellular mechanisms including the purinergic system contribution. Furthermore, the presence and release of nucleotides and dinucleotides during the glaucomatous damage and the maintenance of degradation machinery through ecto-nucleotidase activity are participating in the modulation of the suitable extracellular complex balance. The aim of this study was to investigate the levels of diadenosine tetraphosphate (Ap₄A) and the pattern of ecto-nucleotidase activity expression in glaucomatous retinas during the progress the pathology. Ap₄A levels were analyzed by HPLC in glaucomatous retinas from the DBA/2J mice at 3, 9, 15, and 23 months of age. For that, retinas were dissected as flattened whole-mounts and stimulated in Ringer buffer with or without 59 mM KCl. NPP1 expression was analyzed by RT-PCR and western blot and its distribution was assessed by immunohistochemistry studies examined under confocal microscopy. Glaucomatous mice exhibited Ap₄A values, which changed in stimulated retinas as long as the pathology progressed varying from 0.73?±?0.04 (3 months) to 0.170?±?0.05 pmol/mg retina (23 months). Concomitantly, NPP1 expression was significantly increased (82.15%) in the DBA/2J mice at 15 months. Furthermore, immunohistochemical studies showed that NPP1 labeling was stronger in OPL and IPL labeling tangentially in the vitreal part of the retina and was upregulated at 15 months of age. Our findings demonstrate that Ap₄A decreased levels may be related with exacerbated activity of NPP1 protein in glaucomatous degeneration and in this way contributing to elucidate different mechanisms involved in retinal impairment in glaucomatous degeneration.     

Abnormal metal levels in the primary visual pathway of the DBA/2J mouse model of glaucoma

The purpose of this study was to determine metal ion levels in central visual system structures of the DBA/2J mouse model of glaucoma. We used inductively coupled plasma mass spectrometry (ICP-MS) to measure levels of iron (Fe), copper (Cu), zinc (Zn), magnesium (Mg), manganese (Mn), and calcium (Ca) in the retina and retinal projection of 5-month (pre-glaucomatous) and 10-month (glaucomatous) old DBA/2J mice and age-matched C57BL/6J controls. We used microbeam X-ray fluorescence (μ-XRF) spectrometry to determine the spatial distribution of Fe, Zn, and Cu in the superior colliculus (SC), which is the major retinal target in rodents and one of the earliest sites of pathology in the DBA/2J mouse. Our ICP-MS experiments showed that glaucomatous DBA/2J had lower retinal Fe concentrations than pre-glaucomatous DBA/2J and age-matched C57BL/6J mice. Pre-glaucomatous DBA/2J retina had greater Mg, Ca, and Zn concentrations than glaucomatous DBA/2J and greater Mg and Ca than age-matched controls. Retinal Mn levels were significantly deficient in glaucomatous DBA/2J mice compared to aged-matched C57BL/6J and pre-glaucomatous DBA/2J mice. Regardless of age, the SC of C57BL/6J mice contained greater Fe, Mg, Mn, and Zn concentrations than the SC of DBA/2J mice. Greater Fe concentrations were measured by μ-XRF in both the superficial and deep SC of C57BL/6J mice than in DBA/2J mice. For the first time, we show direct measurement of metal concentrations in central visual system structures affected in glaucoma and present evidence for strain-related differences in metal content that may be specific to glaucomatous pathology.     

DBA/2J Mice Are Susceptible to Diabetic Nephropathy and Diabetic Exacerbation of IOP Elevation

Some pathological manifestations of diabetes in the eye include retinopathy, cataracts and elevated intraocular pressure (IOP). Loss of retinal ganglion cells (RGCs) in non-proliferative stages of diabetic retinopathy and small increases in IOP in diabetic patients has raised the possibility that diabetes affects the development and progression of ocular hypertension and glaucoma. The Ins2^Akita mutation is known to cause diabetes and retinopathy on a C57BL/6J (B6) background by as early as 3 months of age. Here, the impact of the Akita mutation on glaucoma was assessed using DBA/2J (D2) mice, a widely used mouse model of ocular hypertension induced glaucoma. In D2.Ins2^Akita/+ mice, the contribution of diabetes to vascular permeability, IOP elevation, RGC loss, and glaucoma development was assessed. D2.Ins2^Akita/+ mice developed a severe diabetic nephropathy and early mortality between 6–8 months of age. This agrees with previous reports showing that the D2 background is more susceptible to diabetes than the B6 background. In addition, D2.Ins2^Akita/+ mice had vascular leakage, astrocyte reactivity and a significant increase in IOP. However no RGC loss and no anterograde axonal transport dysfunction were found at 8.5 months of age. Therefore, our data show that despite severe diabetes and an increased IOP compared to controls, RGCs do not lose axon transport or degenerate. This may be due to a DBA/2J-specific genetic modifier(s) that could provide novel and important avenues for developing new therapies for diabetic retinopathy and possibly glaucoma.     

Development of in vivo somatic mutation system using antibody against hemoglobin Preparation and use of an anti-hemoglobin antibody for identifying C57BL/6 red cells in artificial mixture of DBA/2 and C57BL/6 red cells

A cellular specific-locus mutation test is described for detecting mutant cells in mammals. The test is based upon the use of specific anti-C57BL/6J mouse hemoglobin antibody that binds hemoglobin “single” (hemoglobin s, present in C57BL/6J mouse) and not hemoglobin “diffuse” (hemoglobin d, present in DBA/2J mouse). Attempts to purify such antibody from pony and rabbit antisera through cross-absorption were unsuccessful. Immunization of LP/J mouse with C57BL/6J hemoglobin produced antiserum that reacted with s hemoglobin but not with d hemoglobin. In a fluorescent antibody technique, this antibody was found to label fixed red blood cells from C57BL/6J mice but not from DBA/2J mice. In a mixture of C57BL/6J and DBA/2J red cells, the C57BL/6J cells could be differentiated by their bright fluorescence from the non-fluorescent DBA/2J cells. Reconstruction experiment with artificial mixtures of DBA/2J and C57BL/6J cells showed that s hemoglobin bearing cells could be detected in DBA/2J red cells at frequencies as small as 0.4×10^?6. Thus, the system is sensitive enough to detect d → s mutation in DBA/2J mice. Amino acid comparison of the globin chains of s and d hemoglobins shows that our antibody can probably detect mutations leading to a substitution of serine or proline by alanine at β²⁰ position and/or a substitution of threonine by alanine at β¹³⁹ position.     

Cyclic AMP accumulation in cerebral cortex tissue from inbred strains of mice.

Large strain differences in neurohumorally induced increases in cyclic AMP can be observed in chopped cerebral cortex of genetically uniform strains of mice. Data from F₁ hybrids of C57BL/6J × DBA/2J and preliminary data from C57BL/6J × SEC/1ReJ suggests dominant transmission by C57 of a chemical factor favoring low accumulation of cyclic AMP in the first cross and recessive transmission of this factor in the second cross. These chemical observations correlate with previously observed transmission of “low active avoidance learning” in the same hybrid strains. Results of determinations of cyclic nucleotide phosphodiesterase activity in supernatant fractions from C57, DBA and the C57 × DBA cross provide a possible explanation for the accumulation differences in those strains.     

Sex-restricted non-Mendelian inheritance of mouse Chromosome 11 in the offspring of crosses between C57BL/6J and (C57BL/6J × DBA/2J)F1 mice

We report on the observation of sex-restricted, non-Mendelian inheritance over a region of mouse Chromosome (Chr) 11, occurring in the offspring of crosses between two commonly used Mus musculus-derived inbred strains, C57BL/6J and DBA/2J. In the surviving backcross progeny of reciprocal matings between (C57BL/6J × DBA/2J)F₁ hybrids and the C57BL/6J parental strain, we observed the preferential appearance of C57BL/6J alleles along a region of Chr 11. The deviation from Mendelian predictions was observed only in female offspring from both reciprocal backcrosses, and not in males from either cross. The sex-specificity of the observed non-Mendelian inheritance points to an explanation based on embryonic or neonatal lethality. Our data add to previously obtained evidence for a Chr 11 locus or loci with sex-specific and allele-specific effects on viability. Received: 19 December 1997 / Accepted: 10 June 1998     

Genetic analysis of barrel field size in the first somatosensory area (SI) in inbred and recombinant inbred strains of mice

We measured the combined area of posterior medial barrel subfield (PMBSF) and anterior lateral barrel subfield (ALBSF) areas in four common inbred strains (C3H/HeJ, A?/J, C57BL?/6J, DBA/2J), B6D2F1, and ten recombinant inbred (RI) strains generated from C57BL/6J and DBA/2J progenitors (BXD) as an initial attempt to examine the genetic influences underlying natural variation in barrel field size in adult mice. These two subfields are associated with the representation of the whisker pad and sinus hairs on the contralateral face. Using cytochrome oxidase labeling to visualize the barrel field, we measured the size of the combined subfields in each mouse strain. We also measured body weight and brain weight in each strain. We report that DBA/2J mice have a larger combined PMBSF/ALBSF area (6.15?±?0.10?mm²,?n?=?7) than C57BL?/6J (5.48?±?0.13?mm²,?n?=?10), C3H/HeJ (5.37?±?0.16?mm²,?n?=?10), and A/J mice (5.04?±?0.09?mm²,?n?=?15), despite the fact that DBA/2J mice have smaller average brain and body sizes. This finding may reflect dissociation between systems that control brain size with those that regulate barrel field area. In addition, BXD strains (average n?=?4) and parental strains showed considerable and continuous variation in PMBSF/ALBSF area, suggesting that this trait is polygenic. Furthermore, brain, body, and cortex weights have heritable differences between inbred strains and among BXD strains. PMBSF/ALBSF pattern appears similar among inbred and BXD strains, suggesting that somatosensory patterning reflects a common plan of organization. This data is an important first step in the quantitative genetic analysis of the parcellation of neocortex into diverse cytoarchitectonic zones that vary widely within and between species, and in identifying the genetic factors underlying barrel field size using quantitative trait locus (QTL) analyses.     

A promising drug candidate for the treatment of glaucoma based on a P2Y6-receptor agonist

Extracellular nucleotides can regulate the production/drainage of the aqueous humor via activation of P2 receptors, thus affecting the intraocular pressure (IOP). We evaluated 5-OMe-UDP(α-B), 1A, a potent P2Y6-receptor agonist, for reducing IOP and treating glaucoma. Cell viability in the presence of 1A was measured using [3-(4, 5-dimethyl-thiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide] (MTT) assay in rabbit NPE ciliary non-pigmented and corneal epithelial cells, human retinoblastoma, and liver Huh7 cells. The effect of 1A on IOP was determined in acute glaucomatous rabbit hyaluronate model and phenol-induced chronic glaucomatous rabbit model. The origin of activity of 1A was investigated by generation of a homology model of hP2Y6-R and docking studies. 1A did not exert cytotoxic effects up to 100 mM vs. trusopt and timolol in MTT assay in ocular and liver cells. In normotensive rabbits, 100 μM 1A vs. xalatan, trusopt, and pilocarpine reduced IOP by 45 vs. 20–30%, respectively. In the phenol animal model, 1A (100 μM) showed reduction of IOP by 40 and 20%, following early and late administration, respectively. Docking results suggest that the high activity and selectivity of 1A is due to intramolecular interaction between Pα-BH³ and C5-OMe which positions 1A in a most favorable site inside the receptor. P2Y6-receptor agonist 1A effectively and safely reduces IOP in normotense, acute, and chronic glaucomatous rabbits, and hence may be suggested as a novel approach for the treatment of glaucoma.     

Biochemical and immunological characterization of genetic variants of phosphoglucose isomerase from mouse

Two electrophoretic variants of phosphoglucose isomerase (PGI) were purified from whole body extracts of DBA/2J and C57BL/6J mice by a substrate-affinity elution from an 8-(6-aminohexyl) amino-ATP-Sepharose column followed by preparative isoelectric focusing. Both PGI variants were shown to be dimers of the same molecular weight, sedimentation coefficient, and K _m for fructose-6-phosphate. The isoelectric points were found to be 8.4 and 8.7 for variants from DBA/2J and C57BL/6J mice, respectively. Differential thermal stability was observed for the two variants in 0.1 m tris-HCl buffer, pH 8.0, at 54 C; the half-lives of the purified PGI from DBA/2J and C57BL/6J mice were shown to be 3.4 and 1.8 min, respectively, under those conditions. Similar differences were observed for the enzyme variants in the crude homogenates. Antisera against PGI from DBA/2J mice were raised in rabbits. The variants from DBA/2J and C57BL/6J mice showed no significant differences in their respective inactivation curves by the antisera. Results of amino acid composition analyses and peptide mappings of the two PGI variants indicate that the genetic variation of this enzyme might result from a single charged amino acid substitution.D. J. C. is a National Institutes of Health Visiting Fellow.     

Measurement of Outflow Facility Using iPerfusion

Elevated intraocular pressure (IOP) is the predominant risk factor for glaucoma, and reducing IOP is the only successful strategy to prevent further glaucomatous vision loss. IOP is determined by the balance between the rates of aqueous humour secretion and outflow, and a pathological reduction in the hydraulic conductance of outflow, known as outflow facility, is responsible for IOP elevation in glaucoma. Mouse models are often used to investigate the mechanisms controlling outflow facility, but the diminutive size of the mouse eye makes measurement of outflow technically challenging. In this study, we present a new approach to measure and analyse outflow facility using iPerfusion^™, which incorporates an actuated pressure reservoir, thermal flow sensor, differential pressure measurement and an automated computerised interface. In enucleated eyes from C57BL/6J mice, the flow-pressure relationship is highly non-linear and is well represented by an empirical power law model that describes the pressure dependence of outflow facility. At zero pressure, the measured flow is indistinguishable from zero, confirming the absence of any significant pressure independent flow in enucleated eyes. Comparison with the commonly used 2-parameter linear outflow model reveals that inappropriate application of a linear fit to a non-linear flow-pressure relationship introduces considerable errors in the estimation of outflow facility and leads to the false impression of pressure-independent outflow. Data from a population of enucleated eyes from C57BL/6J mice show that outflow facility is best described by a lognormal distribution, with 6-fold variability between individuals, but with relatively tight correlation of facility between fellow eyes. iPerfusion represents a platform technology to accurately and robustly characterise the flow-pressure relationship in enucleated mouse eyes for the purpose of glaucoma research and with minor modifications, may be applied in vivo to mice, as well as to eyes from other species or different biofluidic systems.     

The genetics of aryl hydrocarbon hydroxylase induction in mice: A single gene difference between C57BL/6J and DBA/2J

Twenty-one inbred strains of mice were surveyed for inducibility of hepatic aryl hydrocarbon hydroxylase (AHH) activity by the carcinogen 3-methylcholanthrene (MC). In 11 strains given MC, AHH activity increased 1.3- to 5-fold (inducible), whereas ten strains responded with a less than 0.5-fold increase (noninducible). Neither the inducible nor the noninducible class was homogeneous, and in each considerable variation was found in both the basal activity of AHH and the response to MC. Strains DBA/2J and C57BL/6J were chosen to represent the noninducible and inducible classes, respectively. In the crosses (C57BL/6 × DBA/2)F₁ × DBA/2 and (C57BL/6 × DBA/2)F₂, inducibility segregated as a single autosomal dominant gene. The gene symbols Ahh ⁱ and Ahh ⁿ are proposed for the alleles present in C57BL/6J and DBA/2J, respectively. No genetic linkage was found between the Ahh locus and the following loci: b, d, Es-1, Es-3, Gpd-1, Hbb, Id-1, Pgm-1, and sex. Some implications of this work in the study of mammalian enzyme induction and chemically induced carcinogenesis are discussed. There is a positive correlation between AHH inducibility and the development of an inflammatory response to the topical application of the carcinogen 7,12-dimethylbenzanthracene.     

Genetic Differences in Androgen Receptors and in Autoregulation of Testicular Human Chorionic Gonadotropin Binding Sites in the Mouse

Abstract

The autoregulation of testicular human chorionic gonadotropin (hCG) binding sites was studied in two strains of mice known to differ in their endocrine and reproductive characteristics (C57BL/10J and DBA/2J), and in their F₁ progeny (B10D2F₁). Basal hCG binding levels were higher in C57BL/10J than in DBA/2J mice, while B10D2F₁ mice had intermediate levels. Twenty-four h after injection, hCG produced dose-related changes in hCG binding in C57BL/10J and B10D2F₁ mice not observed in DBA/2J mice. However, 72 h after treatment with hCG there was a decrease in hCG binding in all the strains studied. These results suggest the participation of genetic factors in determining basal levels, dose-related changes and temporal response of testicular hCG binding sites to hCG administration. Androgen receptor levels were measured in the same strains of mice. DBA/2J mice had higher receptor levels in the kidney and coagulating gland, and lower levels in the hypothalamus and seminal vesicle when compred to C57BL/10J mice. B10D2F₁ mice had androgen receptor levels similar to those measured in C57BL/10J mice in all tissues studied, with the exception of the coagulating gland, where levels were similar to those observed in DBA/ZJ mice. These observations may indicate the existence of several loci coding for androgen receptors, with only one being expressed per tissue     

The Primary Research on the Gut Microbes in KKAy Mice

In the first part of this paper, gut microbial difference of two genotypes mice was researched. The gut microbial community of type 2 diabetes animal model KKAy mice and normal C57BL/6J mice had clear distinctions in DGGE (denaturing gradient gel electrophoresis) profiles. The pairwise similarity coefficient (C _s ) was only 26–44 % between KKAy and C57BL/6J, but C _s was 82–100 % among same genotypes mice. Thirteen dominant bands were cloned from DGGE profiles to exhibit difference on gut microbial structure further. In the second part of this paper, the influence of hypoglycemic drug Pioglitazone on the gut microbes in KKAy mice was researched by gut microbial diversity analysis and principal component analysis (PCA). The results showed that Pioglitazone reduced the gut microbial diversity slightly and changed gut microbial structure of KKAy mice to that of normal C57BL/6J mice.     

A new locus regulating the expression of the Ldh-2 gene in mouse liver

Patterns of lactate dehydrogenase (LDH) isozymes of tissues from various mouse strains were examined. An interstrain polymorphism for LDH isozymes of liver was established. One phenotype (CBA/Lac and AKR/J mice) yielded a five-banded LDH pattern, another phenotype (DBA/1J, DBA/2J, C57BL/6J and C3H/He) showed a three-banded one. Immunochemical evidence was obtained indicating that differences in the LDH pattern are mainly due to different contents of the B subunit of LDH. Linkage tests indicated that the locus Ldr-2 determining the amounts of the LDH B subunit in mouse liver tissue is located in chromosome 6, 19 ± 4.1 cm away from the earlier described Ldr-1 locus. The effect of locus Ldr-2 is strictly tissue-specific; it is manifest only on days 6–8 after birth.     

Rod photoreceptor ribbon synapses in DBA/2J mice show progressive age-related structural changes

The DBA/2J mouse is a commonly used animal model in glaucoma research. The eyes of DBA/2J mice show severe age-related changes that finally lead to the degeneration of retinal ganglion cells and the optic nerve. Recent electroretinogram studies identified functional deficits, which suggest that also photoreceptor cells are involved in the pathological processes occurring in the DBA/2J mouse retina. In a comparative study, we examined anatomical and molecular changes in the retinae of DBA/2J and C57BL/6 control mice with light and electron microscopy and with PCR analyses. In the retina of the DBA/2J mouse, we found a thinning of the outer plexiform layer, the first synaptic layer in the transfer of visual signals, and age-dependent and progressive degenerative structural changes at rod photoreceptor ribbon synapses. The structural ribbon changes represent a photoreceptor synaptic phenotype that has not yet been described in this animal model of secondary angle-closure glaucoma. Furthermore, genes of the classical complement cascade were upregulated in the photoreceptor cells of aging DBA/2J mice, suggesting a putative link between ribbon synapse degradation and the innate immune system.     

The effect of antisense oligonucleoties specific to the harakiri mRNA on spontaneous and induced defects of mouse preimplantation embryo development

The effect of antisense oligonucleotides specific to mRNA of the proapoptotic gene harakiri (Hrk) on the development of mouse SAMP1 (senescence-accelerated mouse prone) and (C57BL/6J × DBA/2J)F₁ preimplantation embryos cultured in vitro was investigated. The SAMP1 mice are characterized by genetically determined decrease of fertility along with the highly frequent disturbance of embryonic development. Reproduction indices of the (C57BL/6J × DBA/2J) hybrids lie within the normal range. Because of this, preimplantation abnormalities in this line were induced by the action of proapoptotic agent bleomycine. It was demonstrated that antisense inhibition of the Hrk expression had no effect on the frequency of genetically determined abnormalities of early embryonic development in SAMP1 mice. In case of induced abnormalities, addition of oligonucleotides specific to mRNA of proapoptotic Hrk gene influenced the number of abnormalities, and at the same time, improved the quality of survived embryos via increasing the blastocyst hatching.     

Genetic context determines susceptibility to intraocular pressure elevation in a mouse pigmentary glaucoma

Background  

DBA/2J (D2) mice develop an age-related form of glaucoma. Their eyes progressively develop iris pigment dispersion and iris atrophy followed by increased intraocular pressure (IOP) and glaucomatous optic nerve damage. Mutant alleles of the Gpnmb and Tyrp1 genes are necessary for the iris disease, but it is unknown whether alleles of other D2 gene(s) are necessary for the distinct later stages of disease. We initiated a study of congenic strains to further define the genetic requirements and disease mechanisms of the D2 glaucoma.     

Enzyme activities and ethanol preference in mice

Alcohol dehydrogenase and aldehyde dehydrogenase, the two principal enzymes of alcohol metabolism, were assayed in the livers of the inbred mouse strains C57BL/6J and DBA/2J. Previous work has shown that animals of various C57BL substrains prefer a 10% ethanol solution to water in a two-bottle preference test, and that animals of various DBA/2 substrains avoid alcohol. In the present study, C57BL/6J mice were found to have 300% more aldehyde dehydrogenase activity than DBA/2J mice and 30% more alcohol dehydrogenase activity. The F₁ generation is intermediate to the parents in preference for the 10% alcohol solution and is also found to possess intermediate levels of alcohol and aldehyde dehydrogenase activity. These experiments suggest a systematic relationship between the behavioral trait of ethanol preference and the activity of aldehyde dehydrogenase and a similar but much less pronounced relationship with alcohol dehydrogenase.This research was supported by grant GM14547 from the National Institute of General Medical Sciences.     

An interstitial telomere array proximal to the distal telomere of mouse chromosome 13

Lambda clones of mouse DNA from BALB/c and C57BL/10, each containing an array of telomere hexamers, were localized by FISH to a region close to the telomere of Chr 13. Amplification of mouse genomic DNA with primers flanking SSRs within the cloned DNA showed several alleles, which were used to type eight sets of RI strains. The two lambda clones contained allelic versions of the interstitial telomere array, Tel-rs4, which is 495 bp in C57BL/10 and which includes a variety of sequence changes from the consensus telomere hexamer. Comparison of the segregation of the amplification products of the SSRs with the segregation of other loci in an interspecies backcross (C57BL/6JEi × SPRET/Ei) F₁× SPRET/Ei shows recombination suppression, possibly associated with ribosomal DNA sequences present on distal Chr 13 in Mus spretus, when compared with recombination in an interstrain backcross, (C57BL/6J × DBA/J) F₁× C57BL/6J, and with the MIT F₂ intercross. Analysis of recombination in females using a second interstrain backcross, (ICR/Ha × C57BL/6Ha) F₁× C57BL/6Ha, also indicates recombination suppression when compared with recombination in males of the same strains, using backcross C57BL/6Ha × (ICR/Ha × C57BL/6Ha) F₁. Thus, more than one cause may contribute to recombination suppression in this region. The combined order of the loci typed was D13Mit37–D13Mit30–D13Mit148–(D13Rp1, 2, 3, 4, Tel-rs4)–D13Mit53–D13Mit196–D13Mit77–(D13Mit78, 35). Data from crosses where apparently normal frequencies of recombination occur suggest that the telomere array is about 6 map units proximal to the most distal loci on Chr 13. This distance is consistent with evidence from markers identified in two YAC clones obtained from the region. Received: 24 September 1996/Accepted: 20 January 1997     

Characterization of a QTL affecting skeletal size in mice

Abstract Previous work identified a tail length QTL on Chromosome (Chr) 1 in an F₂ population of C57BL/6J × DBA/2J mice. The goals of the present study were to (1) refine the position of this QTL by additional genotyping of samples from the original study; (2) confirm the effect of this QTL by producing a partially congenic strain carrying the C57BL/6J allele against the DBA/2J background; and (3) examine the effect of the QTL on skeletal dimensions. The presence of the QTL was confirmed in a new F₂ population (N = 431) derived from the partially congenic strain, and estimates of its additive effects were similar to those from the original F₂ population (N = 901) in both sexes, i.e., the C57BL/6J chromosomal segment increased tail length, the additive effect (half the difference between homozygotes) being 0.5–0.8 standard deviations. The QTL region was more than halved, relative to that in the previous study, to an 8-cM region between D1Mit30 and D1Mit57. Among a subsample of individuals (N = 30) from the new F₂ population that were not recombinant within the QTL region, there was a significant additive effect of the QTL on the length of the humerus, femur, tibia, mandible, scapula, pelvic girdle, and a tail bone; the direction of the effect was the same as for tail length. No significant effect was found on the number of bones in the tail or on the dimensions of the ulna, skull, or first vertebra.