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: IV. Characterization of 34 Deficiencies

Thirty-four independent nonviable c-locus mutations (types c^al, albino lethal and c^as, albino subvital), derived from radiation experiments, were tested for involvement of nearby markers tp, Mod-2, sh-1, and Hbb: 10, 22, and 2 involved, respectively, none of these markers, Mod-2 alone, and Mod-2 plus sh-1. When classified on this basis, as well as according to developmental stage at which homozygotes die, and by limited complementation results, the 34 independent mutations fell into 12 groups. From results of a full-scale complementation grid of all 435 possible crosses among 30 of the mutations, we were able to postulate an alignment of eight functional units by which the 12 groups fit a linear pattern. Abnormal phenotypes utilized in the complementation study were deaths at various stages of prenatal or postnatal development, body weight, and reduction or absence of various enzymes. Some of these phenotypes can be separated by complementation (e.g., there is no evidence that mitochondrial malic enzyme influences survival at any age); others cannot thus be separated (e.g., glucose-6-phosphatase deficiency and neonatal death).—We conclude that all of the nonviable albino mutations are deficiencies overlapping at c, and ranging in size from <2cM to 6-11 cM. The characterization of this array of deficiencies should provide useful tools for gene-dosage studies, recombinant-DNA fine-structure analyses, etc. Since many of the combinations of lethals produce viable albino animals that resemble the standard c/c type, we conclude (a) that the c locus contains no sites essential for survival, and (b) that viable nonalbino c-locus mutations (c^xv) are the result of mutations within the c cistron. Viable albinos (c^av, the majority of radiation-induced c-locus mutations) may be intracistronic mutations or very small deficiencies.     

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.     

Molecular Genetics of the Brown (B)-Locus Region of Mouse Chromosome 4. I. Origin and Molecular Mapping of Radiation- and Chemical-Induced Lethal Brown Deletions

Over a period of many years, germ-cell mutagenesis experiments using the mouse specific-locus test have generated numerous radiation- and chemical-induced alleles of the brown (b; Tyrp1) locus in mouse chromosome 4. We describe here the origin, maintenance and initial molecular characterization of 28 b mutations that are prenatally lethal when homozygous. Each of these mutations is deleted for Tyrp1 sequences, and each of 25 mutations tested further is deleted for at least one other locus defined by molecular clones previously found to be closely linked to b by interspecific backcross analysis. A panel of DNAs from mice carrying a lethal b mutation and a Mus spretus chromosome 4 was used in the fine structure mapping of these molecularly defined loci. The deletional nature of each of these prenatally lethal mutations is consistent with the hypothesis that the null phenotype at b has an effect only on the quality (color) of eumelanin produced in melanocytes. The resulting deletion map provides a framework on which to build future molecular-genetic and biological analyses of this region of mouse chromosome 4.     

Genotype-temperature interaction inDrosophila melanogaster. I. Viability

Some isogenic strains produced from a natural population at El Prat (Barcelona) were tested at different temperatures for total viability. Their hybrids were also tested at the same temperatures for the same trait. Hybrids proved to be more homcostatic than their isogenic parental strains. More efficient homeostasis in hybrids is suggested to be the cause of increased hybrid superiority with decreasing temperature.Genotype-temperature interaction was always significant for isogenic strains, but never for hybrids. These results are discussed in terms of coadaptation and homeostasis. Some maternal effects have also been detected.     

Chromosome Interactions in DROSOPHILA MELANOGASTER. I. Viability Studies

The nature of fitness interactions is an important, yet unsolved, question in population genetics. We compare the egg-to-adult viability of individuals homozygous for either a second or a third chromosome with the viability of individuals homozygous for both chromosomes simultaneously. On the average, the viability of the two-chromosome homozygotes is somewhat greater than expected assuming that the fitnesses of the single-chromosome homozygotes interact in a multiplicative fashion. This result differs from previous observations that indicate either no significant deviations from the expectation or lower-than-expected average fitnesses for the double homozygotes.     

Origin and Diffusion of the House Mouse in the Mediterranean

The house mouse (Mus musculus), after humans, is the most widespread mammal on earth and one of the worst invasive species for both biological diversity and human health. This ubiquity is the consequence of its strong ecological relationship with humans, namely commensalism. Human activities promote its diffusion by eliminating ecological barriers and by increasing the human environment suitable for this species. This paper deals with recent zooarchaeological data that has helped to decipher the main factors of human evolution involved in the origin of commensal behaviour in the house mouse and in its invasive process throughout the Mediterranean. Understanding this process contributes to our overall knowledge on how human activities modelled mammalian diversity during Holocene. In the Near Eastern core of European Neolithisation, two factors are recognised as the main driving forces to explain the beginning of house mouse commensalism: rise of farming practices (cultivated fields, large scale grain storage, domestication of plants) and human dispersal of domesticated plants through the cultural area of the pre-ceramic Neolithic. These factors increased the attractiveness of the anthropic ecosystem as well as the diffusion vectors of mice by passive transport. Nevertheless, the Neolithisation of the Mediterranean did not promote the house mouse’s invasion of Europe. The commercial and demographic expansions of Phoenicians and Greeks during the last millennium bc were the vectors that allowed the house mouse to overwhelm the ecological barriers that previously prevented its westward invasion of the human environment.     

Dicer Regulates Differentiation and Viability during Mouse Pancreatic Cancer Initiation

miRNA levels are altered in pancreatic ductal adenocarcinoma (PDA), the most common and lethal pancreatic malignancy, and intact miRNA processing is essential for lineage specification during pancreatic development. However, the role of miRNA processing in PDA has not been explored. Here we study the role of miRNA biogenesis in PDA development by deleting the miRNA processing enzyme Dicer in a PDA mouse model driven by oncogenic Kras. We find that loss of Dicer accelerates Kras driven acinar dedifferentiation and acinar to ductal metaplasia (ADM), a process that has been shown to precede and promote the specification of PDA precursors. However, unconstrained ADM also displays high levels of apoptosis. Dicer loss does not accelerate development of Kras driven PDA precursors or PDA, but surprisingly, we observe that mouse PDA can develop without Dicer, although at the expense of proliferative capacity. Our data suggest that intact miRNA processing is involved in both constraining pro-tumorigenic changes in pancreatic differentiation as well as maintaining viability during PDA initiation.     

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.     

Interactions of Polyoma and Mouse DNAs I. Lytic Infection of Bromodeoxyuridine-Prelabeled Mouse Embryo Cells

On CsCl isopycnic centrifugation of the DNA extracted from secondary mouse embryo (ME) cultures grown in the presence of 5-bromodeoxyuridine (BUdR) and 5-fluorodeoxyuridine (FUdR) for 40 h, 10 to 25% of the DNA was found to be unsubstituted, 70 to 80% was bromouracil-hybrid DNA, and 5 to 10% was heavy DNA. These results together with cell number determinations, autoradiography, and Feulgen microspectrophotometry revealed three types of cells in these cultures: (i) 60 to 80% of the cells replicated their DNA once, divided, and then stopped mitotic activity, (ii) 5 to 10% were going through a second round of DNA replication; whereas (iii) 10 to 30% did not replicate DNA during the BUdR-FUdR exposure. After the transfer of these cultures to normal medium (without BUdR-FUdR), up to 20% of the cells resumed DNA synthesis asynchronously within 60 h, but no increase in cell number was observed. BUdR-FUdR-treated cultures, which were infected with polyoma virus in the absence of the thymidine analogues, supported a lytic infection to the same extent as did untreated ME cultures. This was concluded from the similar number of cells, which were induced to synthesize DNA, from the similar replication rate of the viral DNA, from the similar number of cells containing polyoma capsid proteins, and from the similar yields of progeny virus determined by hemagglutination and plaque formation. Thus, BUdR-prelabeled ME cultures are suitable for the investigation of interactions of the polyoma and mouse genomes during the lytic infection.     

Viability of Lettuce Seeds: I. SURVIVAL IN HERMETIC STORAGE

Seeds which show orthodox storage characteristics conform toa common pattern of survival over a wide range of storage conditionswhich can be described by a single equation. Two aspects ofthis are that in a constant environment the life-spans of individualseeds in a population are normally distributed, and that thereis negative linear relationship between log moisture contentand log life-span. However, when orthodox seeds are fully hydratedthey survive much longer than would be predicted by extrapolatingfrom lower moisture contents; but the moisture content at whichthe change occurs has not previously been investigated. In this paper the viability of lettuce seeds was examined aftervarious periods of hermetic storage at different moisture contents,temperatures and initial partial pressures of oxygen. At moisturecontents below 15% the pattern of survival is typical of otherorthodox seeds but above this value the responses change infour ways: instead of being deleterious, oxygen becomes beneficialto survival; instead of life-spans being normally distributed,they become skewed; the relative effect of temperature on decreasinglongevity is slightly diminished; and the decrease in eurvivalperiod with increase in moisture content begins to become lessmarked, so that ultimately, above 20% to 30% moisture content,there is no further decrease in longevity. These results, which indicate substantial physiological changesat about 15% moisture content, are discussed in relation tothe hypothesis, postulated by Villiers, that repair and turnovermechanisms are absent from dry seeds but are activated on hydration. Key words: Lettuce, Lactuca saliva L., Seed viability, Seed storage     

原代小鼠肝脏细胞的高效分离纯化与培养

目的：建立一种能稳定获得高活力和高纯度原代小鼠肝脏细胞的分离、纯化及培养方法。方法：应用改良的Seglen 二步法原位灌注和机械离心分离肝脏细胞,并用改良的高糖DMEM培养基进行培养。台盼蓝拒染法检测接种时肝脏细胞的存活率,倒置显微镜动态观察肝脏细胞形态变化,应用免疫荧光技术对肝脏细胞进行Albumin 染色。结果：每只小鼠可获取肝脏细胞的总产量平均为1.35× 10^6 / g体重,肝脏细胞存活率＞ 90%。倒置显微镜下观察贴壁前肝细胞直径为35.14 滋m± 4.35 滋m,肝脏细胞在接种后3 h基本完成贴壁;肝脏细胞接种后24h,所有肝脏细胞均强阳性表达成熟肝脏细胞标志物Albumin,肝细胞纯度＞ 95%。结论：改良的分离纯化及培养方法能稳定获得高产量、高活率及高纯度的小鼠肝脏细胞。     

Polyamine Metabolism and Osmotic Stress : I. Relation to Protoplast Viability

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine, and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, andVigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.     

Estimation of Fitness Components in DROSOPHILA MELANOGASTER . I. Heterozygote Viability Indices

We examine the assumption of "dominance" with regard to viability of the Cy and Pm marker chromosomes in D. melanogaster . This assumption is often invoked for the extraction of wild-type second chromosomes from natural populations and for the calculation of relative viability indices. Significant genotypic variances for viability are found among both Cy/+_j and Pm/+_i heterozygotes in California and Japanese populations. The magnitude of the Pm/+ _i genotypic variance is substantially less than that of the Cy/+_j heterozygotes (less than one half). Significant reciprocal effects are also found to influence Cy/+_j, Pm/+_i and +_i/+_j viabilities. We conclude that viability indices of heterozygotes based on the Curly method are biased. We suggest that viability indices in the future be expressed relative to the viability of the Cy/Pm genotype (Curly-Plum method) or possibly that of the Pm/+_i genotype (Plum method).     

Mouse liver cell culture. I. Hepatocyte isolation

A method for isolation of mouse liver cells by a two-step perfusion with calcium and magnesium-free Hanks' salt solution followed by a medium containing collagenase is described. Several variations of the commonly used procedure for rat liver cell isolation were quantitatively compared with respect to cell yield and viability. The optimal isolation technique involved perfusion through the hepatic portal vein and routinely produced an average of 2.3 x 10(6) viable liver cells/g body weight. Optimal perfusate collagenase concentration was found to be 100 U of enzyme activity per milliliter of perfusate. Light and electron microscopic evaluation of liver morphology after several steps of the isolation showed distinct morphologic changes in hepatocytes and other liver cells during perfusion. After perfusion with Hanks' calcium- and magnesium-free solution, many hepatocytes exhibited early reversible cell injury. These changes included vesiculation and slight swelling of the endoplasmic reticulum as well as mitochondrial matrix condensation. Subsequent to perfusion with collagenase, the majority of hepatocytes appeared connected to one another only by tight junctional complexes at the bile canaliculi. Multiple evaginations were seen on the outer membrane resembling microville and probably represented the remains of cell-to-cell interdigitations between hepatocytes and sinusoidal lining cells from the space of Disse. The cytoplasmic injury seen after Hanks' perfusion was reversed after collagenase perfusion. After mechanical dispersion, isolated mouse hepatocytes were spherical in shape and existed as individual cells; many (80 to 85%) were binucleated under hase contrast light microscopy. By electron microscopy, cells appeared morphologically similar in cytoplasmic constitution to that seen in intact nonaltered liver cells.     

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.