Analysis of the Albino-Locus Region of the Mouse. II. Mosaic Mutants

Among 119 mutations involving the c locus that were recovered in the course of mouse specific-locus experiments with external radiations, 16 were found in mosaic, or fractional, mutants. The number of additional c-locus fractionals that could have occurred in these experiments and, for a variety of reasons, might not have been clearly identified, probably does not exceed the present number.-There was no evidence for radiation induction of the fractionals, and even those occurring in the irradiated groups may thus be assumed to be of spontaneous origin. Since only two mutations in the control groups were found in whole-body mutants, it appears that the bulk of spontaneous c-locus mutations are fractionals.-None of the mutations recovered in fractional mutants was homozygous lethal; 25% were viable intermediate alleles, and the remainder were albino-like mutants, all viable except for one subvital and one not tested.-Genetic tests of the fractionals indicated no major selection against the new mutations, either gametically or in the progeny.-For the group of fractionals as a whole, about one-half of the germinal tissue carried the mutation, indicating that the fractionals came from an overall blastomere population that was one-half mutant. Such a population could result from mutation in one strand of the gamete DNA, in a daughter chromosome derived from pronuclear DNA synthesis of the zygote, or in one of the first two blastomeres prior to replication. Since the mouse embryo does not stem from all of the cleavage products of the zygote, the frequency of fractionals observed underestimates the frequency of mutational events that result in two types of blastomeres.     

Analysis of the Albino-Locus Region of the Mouse. I. Origin and Viability

Numerous specific-locus experiments designed to test the mutagenic effect of external radiation have yielded, in over 3,600,000 animals observed, altogether 119 presumed mutations involving the c locus. Of these, 55 were viable and albino (cav), 13 were viable and of various intermediate pigment types (cxv), four were subvital (cas and cxs), seven were neonatally lethal albinos (cal), 28 prenatally lethal albinos (cal); 12 died untested. All of the prenatally lethal and at least one of the neonatally lethal c-locus mutations (cal classes) are probably deficiencies that we have analyzed extensively in other experiments. Since absence of the locus mimics albino in phenotype, the intermediates (cxv and cxs groups) probably resulted from intragenic changes. The class of viable albino mutants (cav) might include, in addition to intragenic changes, some extremely small deficiencies. --The effects on viability of c-locus lethals (cal's) in heterozygous condition are not drastic enough to be perceived in stocks of mixed genetic background except in the case of the two longest known deficiencies and a few others. --Analysis of the relation between radiation treatment and type of c-locus mutants obtained shows that the relative frequency of viable mutations, for each germ-cell type, is greater for low-LET than for neutron irradiation; however, the difference for any individual cell type is not significant. The majority (66.7%) of mutations derived from X- or gamma-ray irradiated spermatogonia are viable, and the proportion of "intermediates" among these viables is similar to that among presumed spontaneous c-locus mutations. No significant dose-rate effect on the proportion of lethals could be demonstrated within the set of mutants induced by low-LET irradiation of spermatogonia. Although sets from other germ-cell stages are too small for statistical tests, the results for oocytes are similar, as far as they go. Furthermore, most of the c-locus mutations induced in spermatogonia, even by high-dose-rate X-ray or gamma irradiation, are of a type most likely to result from single-tract events (62% cxv, cxs, and cav; plus 16% presumed deficiencies not involving the closest marker). These results support the view that most of the reduction in mutation frequency at low dose rates is not due to a change in relative proportion of two-track and one-track ionizing events.     

Analysis of the Albino-Locus Region of the Mouse. III. Time of Death of Prenatal Lethals

The stage at which homozygotes die was determined for 28 mutations (general symbol c*) at the albino (c) locus, of which 26 had earlier been found to be probably prenatally lethal. Within each of the mutant stocks, the uterine contents of c*/c(ch) females, made pregnant either by c*/c(ch) males ("Ex" series) or by c(ch)/c(ch) males ("Co" series), were examined between 13 and 17 days postconception. Altogether, 743 females were dissected and 7197 corpora lutea (representing ovulations) counted. In selected stocks, an additional 40 and 13 females were dissected on days seven or nine, respectively.-In each of the 26 presumed prenatally lethal mutants, there was a deficiency of living fetuses in the Ex, as compared with the Co, group. Overall, this deficiency was 23.6% (expectation, 25% c*/c*). All meaningful excesses were in numbers either of moles (death shortly before, during, or just after implantation), or of early preimplantation losses. Homozygotes in none of the mutant stocks die between days nine and 19 postconception. Of 24 c-locus mutants known to be deficiencies since they lack the closely linked Mod-2, 13 clearly kill before implantation, ten around implantation, and one neonatally. The c and Mod-2 loci and the region between them are not needed for intrauterine survival.-There are indications that the distinction between early-preimplantation death and implantation death may, in a general way, be related to length of the deficiency.     

Essential Genes and Deficiencies in the UNC-22 IV Region of CAENORHABDITIS ELEGANS

Five formaldehyde-induced deficiencies that uncover unc-22 IV, a gene affecting muscle structure in the nematode Caenorhabditis elegans were isolated and positioned. The largest deficiency, sDf2, extends in both directions from unc-22 and is approximately 1.0–2.0 map units in length. The other four deficiencies, sDf7, sDf8, sDf9 and sDf10, are all smaller than sDf2 and are located within the region uncovered by this deficiency. Thirty-seven ethyl methanesulfonate-induced lethal and sterile mutations linked to unc-22 were isolated and tested for complementation with sDf2. Nineteen lethal mutations failed to complement sDf2. Sixteen of these were further positioned by recombination mapping and also by deficiency mapping with sDf7, sDf8, sDf9 and sDf10. These sixteen mutations define 11 new essential genes in this region. Eight of the genes lie in a 0.9-map unit interval to the left of unc-22, whereas the three remaining genes lie in a region of about 0.2 map units to the right of unc-22. We believe that two of the essential genes identified in this study, let-56 and let-52, are the adjacent genes on either side of unc-22. The lethal mutations exhibit a wide range of terminal phenotypes: from first stage larva to sterile adult.     

人及小鼠钠通道SCN3A基因的启动子及其上游调控区的分析

为了比较研究人与小鼠SCN3A基因的启动子及其上游调控区的特征,采用5′-Full RAGE方法对人及小鼠SCN3A基因的转录起始点进行了准确定位,通过序列测定及对比分析证明:确定人和小鼠SCN3A基因的转录起始点均为"A",人SCN3A基因转录起始点位于翻译起始点上游约27 kb处,而小鼠位于翻译起始点上游约31 kb处.人SCN3A基因5′非翻译区存在两个5′非翻译外显子,而小鼠只有一个5′非翻译外显子.人和小鼠SCN3A基因核心启动子区(-80～+70)的同源率高达96.0%,存在相同的启动子核心元件,BRE/和TATA;在-400至+200区段内预测到人存在而小鼠不存在的转录因子有PHR1、GATA-1、FOXN2、NF-及AP-4,小鼠存在而人不存在的转录因子Sp、Sp3及GBF.人和小鼠SCN3A启动子区特征的异同将为进一步研究该基因在人和小鼠的表达调控机制提供重要线索.     

Transgenic Mouse Model of Hemifacial Microsomia: Cloning and Characterization of Insertional Mutation Region on Chromosome 10

The 643 transgenic mouse line carries an autosomal dominant insertional mutation that results in hemifacial microsomia (HFM), including microtia and/or abnormal biting. In this paper, we characterize the transgene integration site in transgenic mice and preintegration site of wildtype mice. The locus, designated Hfm (hemifacial microsomia-associated locus), was mapped to chromosome 10, B1-3, by chromosome in situ hybridization. We cloned the transgene insertion site from the transgenic DNA library. By using the 5′ and 3′ flanking sequences, the preintegration region was isolated. The analysis of these regions showed that a deletion of at least 23 kb DNA occurred in association with the transgene integration. Evolutionarily conserved regions were detected within and beside the deleted region. The result of mating between hemizygotes suggests that the phenotype of the homozygote is lethality in the prenatal period. These results suggest that the Hfm locus is necessary for prenatal development and that this strain is a useful animal model for investigating the genetic predisposition to HFM in humans.     

小鼠PD-1基因启动子克隆鉴定及转录调控分析

PD-1分子是一种重要的免疫调控因子,目前对其自身表达调控尚未有系统的研究.对PD-1启动子区域进行分析,克隆构建了含PD-1基因上游约2kb范围内含4种不同长度调控序列的双荧光素酶表达载体.通过FACS检测发现,小鼠T淋巴瘤EL4细胞稳定表达PD-1,而骨髓瘤Sp2/0-Ag细胞则在佛波酯(phorbol 12-myristate 13-acetate,PMA)和离子霉素(ionomycin,IO)诱导后才表达PD-1.4种双荧光素酶报告系统在上述两种细胞中检测PD-1启动子各区段活性显示,-227～+49bp区域含有PD-1核心启动子,上游-1127～-716bp含有较强正性调节元件,而在-1685～-1128bp、-715～-228bp两个区域含有负性调节元件,这种正负调控区交错的启动子活性现象说明了PD-1基因表达调控的复杂性,这些结果为PD-1基因的表达调控提供了结构基础和依据.     

人及小鼠钠通道SCN3A基因的启动子及其上游调控区的分析

为了比较研究人与小鼠SCN3A基因的启动子及其上游调控区的特征,采用5′-Full RACE方法对人及小鼠SCN3A基因的转录起始点进行了准确定位,通过序列测定及对比分析证明：确定人和小鼠SCN3A基因的转录起始点均为“A”,人SCN3A基因转录起始点位于翻译起始点上游约27 kb处,而小鼠位于翻译起始点上游约31 kb处．人SCN3A基因5′非翻译区存在两个5′非翻译外显子,而小鼠只有一个5′非翻译外显子．人和小鼠SCN3A基因核心启动子区(-80 ～+70)的同源率高达96.0%,存在相同的启动子核心元件,BRE/和TATA;在-400至+200区段内预测到人存在而小鼠不存在的转录因子有PHR1、GATA-1、FOXN2、NF-1及AP-4,小鼠存在而人不存在的转录因子Sp、Sp3及GBF．人和小鼠SCN3A启动子区特征的异同将为进一步研究该基因在人和小鼠的表达调控机制提供重要线索．     

Potassium Transport in Corn Roots : IV. Characterization of the Linear Component

A detailed examination was conducted on the linear, or first-order kinetic component for K⁺(⁸⁶Rb⁺) influx into root segments of both low- and high-salt grown corn seedlings (Zea mays [A632 × Oh 43]). In tissue from both low- and high-salt grown roots, replacement of Cl⁻ in the uptake solution by either SO₄²⁻, H₂PO₄⁻, or NO₃⁻ caused a significant (50-60%) and specific inhibition of the linear component of K⁺ influx. The anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid, was found to abolish saturable Cl⁻ influx in corn roots while causing a significant (50-60%) and specific inhibition of the linear K⁺ uptake system; this inhibition was identical to that observed when Cl⁻ was replaced by other anions in the K⁺ uptake solution. Additionally, the quaternary ammonium cation, tetraethylammonium, which has been shown to block K⁺ channels in nerve axons, also caused a dramatic (70%) and specific inhibition of the linear component of K⁺ influx, but this was obtained only in high-salt roots. The reasons for this difference are discussed with respect to the differing abilities of low- and high-salt roots to absorb tetraethylammonium.

Our present results indicate that the linear component of K⁺ influx may occur by a passive process involving transmembrane K⁺ channels. Fluxes through these K⁺ channels may be partly coupled to a saturating Cl⁻ influx mechanism.

     

Molecular Genetic Analysis of the DILUTE-SHORT EAR ( d-se) Region of the Mouse

Genes of the dilute-short ear (d-se) region of mouse chromosome 9 comprise an array of loci important to the normal development of the animal. Over 200 spontaneous, chemically induced and radiation-induced mutations at these loci have been identified, making it one of the most genetically well-characterized regions of the mouse. Molecular analysis of this region has recently become feasible by the identification of a dilute mutation that was induced by integration of an ecotropic murine leukemia virus genome. Several unique sequence cellular DNA probes flanking this provirus have now been identified and used to investigate the organization of wild-type chromosomes and chromosomes with radiation-induced d-se region mutations. As expected, several of these mutations are associated with deletions, and, in general, the molecular and genetic complementation maps of these mutants are concordant. Furthermore, a deletion breakpoint fusion fragment has been identified and has been used to orient the physical map of the d-se region with respect to the genetic complementation map. These experiments provide important initial steps for analyzing this developmentally important region at the molecular level, as well as for studying in detail how a diverse group of mutagens acts on the mammalian germline.     

The Genetics of a Small Autosomal Region of DROSOPHILA MELANOGASTER Containing the Structural Gene for Alcohol Dehydrogenase. I. Characterization of Deficiencies and Mapping of ADH and Visible Mutations

The position of the structural gene coding for alcohol dehydrogenase (ADH) in Drosophila melanogaster has been shown to be within polytene chromosome bands 35B1 and 35B3, most probably within 35B2. The genetic and cytological properties of twelve deficiencies in polytene chromosome region 34--35 have been characterized, eleven of which include Adh. Also mapped cytogenetically are seven other recessive visible mutant loci. Flies heterozygous for overlapping deficiencies that include both the Adh locus and that for the outspread mutant (osp: a recessive wing phenotype) are homozygous viable and show a complete ADH negative phenotype and strong osp phenotype. These deficiencies probably include two polytene chromosome bands, 35B2 and 35B3.     

Genetic Organization of the agouti Region of the Mouse

The agouti locus on mouse chromosome 2 acts via the hair follicle to control the melanic type and distribution of hair pigments. The diverse phenotypes associated with various agouti mutations have led to speculation about the organization of the agouti locus. Earlier studies indicated that two presumed agouti alleles, lethal yellow (Ay) and lethal light-bellied nonagouti (ax), are pseudoallelic. We present genetic data showing probable recombination between Ay and three agouti mutations (at, a, and ax), which suggest that Ay is a pseudoallele of the agouti locus. The close linkage of an endogenous ecotropic murine leukemia provirus, Emv-15, to Ay provides a molecular access to genes at or near the agouti locus. However, previous studies suggested that the Emv-15 locus can recombine with some agouti alleles and therefore we analyzed mice from recombinant inbred strains and backcrosses to measure the genetic distance between various agouti alleles and the Emv-15 locus. Our data indicate that the Emv-15 locus is less than 0.3 cM from the agouti locus. These experiments provide a conceptual framework for initiating chromosome walking experiments designed to retrieve sequences from the agouti locus and give new insight into the genetic organization of the agouti region.     

Nuclear membranes from mammalian liver : IV. Characterization of membrane-attached DNA

DNA associated with nuclear membranes isolated from liver tissue of mice and rats (sucklings and partially hepatectomized adults) has been analysed and directly demonstrated by electron microscopy using spreading techniques. The sensitivity of this DNA-membrane association to DNAse, to 4 M CsCl-centrifugation, urea, and to detergent has been examined and compared with that of ‘microsomal DNA’. The DNA has been purified from nuclear membrane fractions, and the purity and molecular size distribution of the preparations has been determined. The characteristics of this DNA with respect to buoyant density, melting behaviour, content of repetitive sequences, nucleotide composition, molecular configuration, and turnover and labelling kinetics with various precursors (thymidine, deoxycytidine, phosphate) have been examined and compared with the corresponding properties of DNA from whole nuclei and other nuclear subfractions. Most properties of membrane DNA are identical or similar to those of bulk nuclear DNA. It is, however, enriched in satelite DNA and other repetitive sequences to a moderate extent and differs from it in its replication rate and time. The results reflect the close relationship between the nuclear envelope and (constitutive) heterochromatin, but also indicate that membrane binding is not restricted to this material. The data speak against a preferential localization of replicating points in the nuclear membrane DNA, as well as against an initiation of replication at the nuclear envelope.     

Biophysical and Biochemical Characterization of Lymphocytic Choriomeningitis Virus: IV. Strain Differences

Biological, biochemical, and biophysical properties of three lymphocytic choriomeningitis (LCM) virus strains were compared. The biological property examined was the concentration range of virus which would, when injected into neonates, cause a carrier state. The dosage range for the CA1371 and Traub strains was found to be as broad as the limits examined (5 to 100 ld(50) units/mouse). The WCP strain, however, would only produce carriers within a 3 to 5 ld(50) range. The biochemical properties examined were the growth rates in tissue culture and the effect of varying the input ratio of virus to cells. With identical input ratios, the Traub strain reached a peak titer 32 hr after infection. The CA1371 and WCP strain reached their peaks at the 40th hr. With a 10-fold decrease in the amount of CA1371 virus per cell, peak titer (as high as in the above experiments) was not obtained until 56 hr postinfection. The biophysical properties examined were stability in density gradients and inactivation rates at 4C. In potassium tartrate gradients, full recovery of the CA1371 and WCP strain could be achieved. However, inactivation kinetics showed that only the CA1371 strain was much more stable than the Traub-LCM. The realization that marked differences in LCM strains exist is discussed in relation to certain inconsistencies in the literature.     

Maternal-Zygotic Lethal Interactions in DROSOPHILA MELANOGASTER: The Effects of Deficiencies in the Zeste-White Region of the X Chromosome

The possibility that essential loci in the zeste-white region of the Drosophila melanogaster X chromosome are expressed both maternally and zygotically has been tested. Maternal gene activity was varied by altering gene dose, and zygotic gene activity was manipulated by use of position-effect variegation of a duplication. Viability is affected when both maternal and zygotic gene activity are reduced, but not when either maternal or zygotic gene activity is normal. Tests of a set of overlapping deficiencies demonstrate that at least three sections of the zeste-white region yield maternal zygotic lethal interactions. Single-cistron mutations at two loci in one of these segments have been tested, and maternal heterozygosity for mutations at both loci give lethal responses of mutant-duplication zygotes. Thus, at least four of the 13 essential functions coded in the zeste-white region are active both maternally and zygotically, suggesting that a substantial fraction of the genome may function at both stages. The normal survival of zygotes when either maternal gene expression or zygotic gene expression is normal, and their inviability when both are depressed, suggests that a developmental stage exists when maternally determined functions and zygotically coded functions are both in use.     

Structure of the Mouse Mammary Tumor Virus: Characterization of Bald Particles

The polypeptide, antigenic, and morphological structure of the mouse mammary tumor virus was studied following protease digestion of intact virions. Intact, untreated virions (rho = 1.17 g/ml) had characteristic envelope spikes, five major polypeptides, and were precipitated by antisera against gp52. Two of the major polypeptides, with molecular weights of 52,000 (gp52) and 36,000 (gp36), had carbohydrate moieties. Protease treatment resulted in spikeless, "bald" particles (rho = 1.14 g/ml), which had altered surface antigenicity and which contained neither gp52 nor gp36. These data indicated that gp52 and gp36 were on the viral envelope. Bald particles retained a 28,000 dalton polypeptide (p28) which was proposed as the major internal polypeptide.