Gametic imprinting and the manifestation of the fused gene in the house mouse

The phenomenon of gametic imprinting in mammals has raised developmentally relevant questions concerning the manifestation and inheritance of genes with variable penetrance. The dominant fused (Fu) gene located on chromosome 17 is one of the few good cases demonstrating the phenomenon in mice. The Fu mutation has a maternal effect. We have previously shown that the t12 haplotype significantly lowers the penetrance of Fu in female Fu/t12 offspring. Results of reciprocal matings of the heterozygotes for Fu indicated that the Fu of maternal origin has a lowered level of penetrance. The dominant suppressors located outside chromosome 17, in contrast to t12 residing in it, had stronger effects on the manifestation of Fu, decreasing its penetrance to 8-17%. Experimental evidence is presented that the pathway via which Fu passes to the zygote nucleus during gametogenesis through successive generations has a marked effect on its penetrance. Based on this evidence, patterns of genetic imprinting are described. A survey of genetic imprinting allowed us to distinguish two developmental phases, gametic and zygotic. The hypothesis for the gametic phase of the development of multicellular organisms suggests that it proceeds from initialization, a process thought to ensure the freeing of chromosomes from redundant epigenetic information and their preparation for the consecutive developmental cycle.     

Analysis of hypotheses and simulation of selection for a suppressor affecting the manifestations of the fused gene in mice

The t12 haplotype decreases sharply manifestation of fused gene in mice. The selection for increasing the penetrance of fused gene was made in Fu/t12 stock. The different computer models of selection process were compared. The hypothesis giving the best coincidence with experimental data supposes the presence of a semidominant gene suppressing the effect of t12 on Fu gene in chromosome 8.     

Gametic imprinting and expression of the gene Fused in the house mice

The discovery of the gametic imprinting phenomena in mammals makes it possible to have a new look at some facts concerning the expression and inheritance of genes with variable penetrance. Fused (Fu) is a dominant mutation located on chromosome 17, one of the few examples uses to demonstrate gametic imprinting in mice. This mutation has maternal effect connected with decrease in its penetrance. It was shown that t12 haplotype significantly reduces penetrance of the Fu in the progeny of Fu/t12 females. The results of reciprocal crosses of heterozygotes for t12 haplotypes indicate that penetrance of maternal Fu gene is reduced. Far more strong influence on the fused penetrance have the dominant suppressors, located beyond the chromosome 17. The penetrance of the fused gene decreases in that case up to 8-17%. Results of the experiments show strong influence of gametic pathway on penetrance of the gene by which the gene is transmitted to the next generation. The results also made it possible to describe the regularities of gametic imprinting. This phenomenon clearly indicates the existence of gametic and zygotic ontogenetic phases. According to the hypothesis proposed gametic phase of ontogenesis in mammals starts after initialization, i. e. after a process of chromosome erasing from epigenetic information and preparing to enter the next ontogenetic cycle.     

Genomic organization of the S-locus region of Brassica

To gain some insights into the structure of the S-locus and the mechanisms that have kept its diversity, a 75-kb genomic fragment containing the self-incompatibility (S) locus region was isolated from the S12-haplotype of Brassica rapa and compared with those of other S-haplotypes. The region around the S determinant genes was highly polymorphic and filled with S-haplotype-specific intergenic sequences. The diverse genomic structure must contribute to the suppression of recombination at the S-locus.     

Chromosome segregation in mice heterozygous for Robertsonian translocations. I. The effect of mutation in chromosome 17 on the disjunction of homologs in females

The effects of mutations on chromosome 17 upon the segregation of the metacentric and acrocentric homologues in the progeny of female mice heterozygous for Robertsonian translocations Rb(8.17) 1Iem and Rb(16.17) 7Bnr were studied. Genetic analysis indicated that the ratio of Rb to non-Rb (normal karyotype) progeny from mothers heterozygous for mutations tf, qk, t12 were not significantly different from 1:1 expected. Introduction of mutations T, Ki, Fu, t6 into the female genotype caused strong distortion of segregation and an increase in the proportion of progeny with normal karyotype (65-70%). From the data on embryonic mortality and cytogenetic observations, it is concluded that distortion of equal transmission arises before MII of meiosis. Consequently, preferential distribution of the metacentric chromosome into the polar body during the first meiotic division is relevant to the segregation distortion observed.     

Characterization of novel partial t-haplotypes tM1, tM2, tM3, tM4]

The results of genetic analysis of the effects of four novel partial mouse TM-haplotypes are presented in this work. Fertility and viability of tM/tM homozygotes and tM/t6 compounds, transmission ratio distortion (trd) in males heterozygous for tM, suppression of recombination and taillessness effects were studied. Three novel t-haplotypes tM1,2,4 are viable and heterozygous for these haplotypes males T/tM show low trd (20-30%). Comparison of these data and the t6-haplotype structure suggests that the tM1,2,4-haplotypes were derived as a result of recombination events in the non-inverted T-complex fragment located between two inversions. The tM3-haplotype is semilethal and heterozygous T/tM3 males show the trd equal to that of t6-heterozygotes. Homozygous tM3/tM3 and tM3/t6 male compounds are fertile or subfertile. Potential recombination ways of derivation of tM3 are discussed.     

小鼠成纤维细胞凋亡过程中P53与bcl-2表达的时序性

为探讨细胞凋亡过程中,ｂｃｌ－２与Ｐ５３,这两种凋亡关键性基因的相互关系,用５－氟尿嘧啶（５－Ｆｕ）诱导小鼠正常与恶性转化的成纤维细胞的凋亡,观察了这两种基因在凋亡过程中表达变化的时序．经流式细胞计检测,这两种细胞在５－Ｆｕ作用２４ｈ均出现了凋亡峰,细胞存活率随之下降,ＤＮＡ电泳显现梯状断裂．Ｎｏｒｔｈｅｒｎ杂交结果显示,在５－Ｆｕ作用６ｈ后两种细胞Ｐ５３ｍＲＮＡ水平已明显增高,而ｂｃｌ－２ｍＲＮＡ水平则在作用１２ｈ方明显降低．Ｐ５３上调与ｂｃｌ－２下调的明显时序性,说明Ｐ５３具备作为ｂｃｌ－２基因负调控转录因子的条件．由此,为进一步了解凋亡过程的ｂｃｌ－２基因下调机理提供了线索     

The mutated S1-haplotype in sour cherry has an altered S-haplotype-specific F-box protein gene

Gametophytic self-incompatibility (GSI) is an outcrossing mechanism in flowering plants that is genetically controlled by 2 separate genes located at the highly polymorphic S-locus, termed S-haplotype. This study characterizes a pollen part mutant of the S(1)-haplotype present in sour cherry (Rosaceae, Prunus cerasus L.) that contributes to the loss of GSI. Inheritance of S-haplotypes from reciprocal interspecific crosses between the self-compatible sour cherry cultivar Ujfehértói Fürt?s carrying the mutated S(1)-haplotype (S(1)'S(4)S(d)S(null)) and the self-incompatible sweet cherry (Prunus avium L.) cultivars carrying the wild-type S(1)-haplotype revealed that the mutated S(1)-haplotype confers unilateral incompatibility with a functional pistil component and a nonfunctional pollen component. The altered sour cherry S(1)-haplotype pollen part mutant, termed S(1)', contains a 615-bp Ds-like element within the S(1)-haplotype-specific F-box protein gene (SFB(1)'). This insertion generates a premature in-frame stop codon that would result in a putative truncated SFB(1) containing only 75 of the 375 amino acids present in the wild-type SFB(1). S(1)' along with 2 other previously characterized Prunus S-haplotype mutants, S(f) and S(6m), illustrate that mobile element insertion is an evolutionary force contributing to the breakdown of GSI.     

Chromosome segregation in mice heterozygous for Robertsonian translocations. II. Changes in the structure of homologs as a cause of abnormal disjunction in females

It was demonstrated that mutations T, Fu, Ki, t6 of chromosome 17 cause preferential transmission of the acrocentric homologues to the progeny from female Rb heterozygotes. The results indicate that the effects of these mutations on segregation are restricted to the Robertsonian translocations involving chromosome 17. Substitution of the parts of chromosome 17 distal or proximal to the T-locus did not alter the effect, of this chromosome on the transmission rate of the homologue. The transmissions effects of these mutations, whether cis or trans with Rb, were the same. It was observed that mothers Rb7/T43H transmitted the chromosome with the reciprocal translocation T43H to 70.9% of their progeny. Data were obtained supporting the idea that structural changes of the chromosomes caused by mutations affect segregation of the homologues in Rb heterozygous females. The possible mechanism of this influence is discussed.     

Analysis of the S-locus structure in Prunus armeniaca L. Identification of S-haplotype specific S-RNase and F-box genes

The gametophytic self-incompatibility (GSI) system in Rosaceae has been proposed to be controlled by two genes located in the S-locusan S-RNase and a recently described pollen expressed S-haplotype specific F-box gene (SFB). However, in apricot (Prunus armeniaca L.) these genes had not been identified yet. We have sequenced 21kb in total of the S-locus region in 3 different apricot S-haplotypes. These fragments contain genes homologous to the S-RNase and F-box genes found in other Prunusspecies, preserving their basic gene structure features and defined amino acid domains. The physical distance between the F-boxand the S-RNase genes was determined exactly in the S ₂-haplotype (2.9kb) and inferred approximately in the S ₁-haplotype (< 49kb) confirming that these genes are linked. Sequence analysis of the 5 flanking regions indicates the presence of a conserved region upstream of the putative TATA box in the S-RNase gene. The three identified S-RNase alleles (S ₁, S ₂ and S ₄) had a high allelic sequence diversity (75.3 amino acid identity), and the apricot F-box allelic variants (SFB₁, SFB₂ and SFB₄) were also highly haplotype-specific (79.4 amino acid identity). Organ specific-expression was also studied, revealing that S ₁- and S ₂-RNases are expressed in style tissues, but not in pollen or leaves. In contrast, SFB ₁ and SFB ₂ are only expressed in pollen, but not in styles or leaves. Taken together, these results support these genes as candidates for the pistil and pollen S-determinants of GSI in apricot.     

The in vivo and in vitro transmission frequencies of the tw5-haplotype in mice

The recessive tw5-haplotype, a complete haplotype, is transmitted by heterozygous male mice at very high frequencies (greater than 0.90) in normal matings. The present studies were undertaken to determine the effects of delayed matings and in vitro fertilizations on this phenotypic expression. Males carrying the tw5-haplotype (+/tw5) were first tested for their frequencies of transmission of the mutant 17th chromosome in both normal and delayed matings. Spermatozoa obtained from these same males were then used to fertilize eggs in vitro. The in vivo and in vitro transmission frequencies were found to be statistically equivalent in all types of inseminations. An in vitro fertilization time course study showed that the same percentages of eggs are fertilized by tw5-bearing spermatozoa when the gametes are coincubated for either 2 or 6 h. The data lead to the conclusion that the transmission frequency of the tw5-haplotype is not affected either by the length of time elapsing between insemination and fertilization or by the environment in which fertilization occurs.     

Transfection of DHFR- and DHFR+ mammalian cells using methotrexate-resistant mutants of mouse dihydrofolate reductase

Site-directed mutagenesis was used to generate mutants of mouse dihydrofolate reductase more resistant to methotrexate than the wild type enzyme. The mutant genes were used to transfect either DHFR- or DHFR+ cell lines. These mutants, as well as the wild type gene, were able to confer methotrexate resistance to DHFR- CHO cells. The number of selected colonies decreased with increased concentrations of methotrexate. The number of colonies observed at 10 microM methotrexate is correlated with the Ki(MTX) of the enzyme: the higher the Ki, the higher the number of colonies for the corresponding mutant. In contrast, the transfection of DHFR+ cells gave a few numbers of colonies not different for the wild type and the mutants.     

The transmission ratio distortion of the th2-haplotype in vivo and in vitro

The th2-haplotype is transmitted at low frequencies (less than 0.30) by +/th2 males in normal matings. In the studies described here, the transmission frequency of the th2-haplotype from Rb7/th2 males was determined for normal and delayed matings and in vitro inseminations. The data show the transmission frequency from the two in vivo inseminations to be less than 0.30 and to be statistically equivalent. However, the in vitro transmission frequency (0.80) is significantly greater than either of the in vivo frequencies. The results show that the environment in which fertilization occurs affects the transmission frequency of this specific t-haplotype significantly.     

Self-compatibility of two apricot selections is associated with two pollen-part mutations of different nature

Loss of pollen-S function in Prunus self-compatible mutants has recently been associated with deletions or insertions in S-haplotype-specific F-box (SFB) genes. We have studied two self-compatible cultivars of apricot (Prunus armeniaca), Currot (S(C)S(C)) and Canino (S(2)S(C)), sharing the naturally occurring self-compatible (S(C))-haplotype. Sequence analysis showed that whereas the S(C)-RNase is unaltered, a 358-bp insertion is found in the SFB(C) gene, resulting in the expression of a truncated protein. The alteration of this gene is associated with self-incompatibility (SI) breakdown, supporting previous evidence that points to SFB being the pollen-S gene of the Prunus SI S-locus. On the other hand, PCR analysis of progenies derived from Canino showed that pollen grains carrying the S(2)-haplotype were also able to overcome the incompatibility barrier. However, alterations in the SFB(2) gene or evidence of pollen-S duplications were not detected. A new class of F-box genes encoding a previously uncharacterized protein with high sequence similarity (approximately 62%) to Prunus SFB proteins was identified in this work, but the available data rules them out of producing S-heteroallelic pollen and thus the cause of the pollen-part mutation. These results suggest that cv Canino has an additional mutation, not linked to the S-locus, which causes a loss of pollen-S activity when present in pollen. As a whole, these findings support the proposal that the S-locus products besides other S-locus independent factors are required for gametophytic SI in Prunus.     

The role of GLI2-ABCG2 signaling axis for 5Fu resistance in gastric cancer

Gastric cancer is a leading cause of cancer-related mortality worldwide, and options to treat gastric cancer are limited. Fluorouracil(5Fu)-based chemotherapy is frequently used as a neoadjuvant or an adjuvant agent for gastric cancer therapy. Most patients with advanced gastric cancer eventually succumb to the disease despite the fact that some patients respond initially to chemotherapy. Thus, identifying molecular mechanisms responsible for chemotherapy resistance will help design novel strategies to treat gastric cancer. In this study, we discovered that residual cancer cells following 5Fu treatment have elevated expression of hedgehog(Hg) target genes GLI1 and GLI2, suggestive of Hh signaling activation. Hh signaling, a pathway essential for embryonic development, is an important regulator for putative cancer stem cells/residual cancer cells. We found that high GLI1/GLI2 expression is associated with some features of putative cancer stem cells, such as increased side population. We demonstrated that GLI2 knockdown sensitized gastric cancer cells to 5Fu treatment, decreased ABCG2 expression, and reduced side population. Elevated GLI2 expression is also associated with an increase in tumor sphere size, another marker for putative cancer stem cells. We believe that GLI2 regulates putative cancer stem cells through direct regulation of ABCG2. ABCG2 can rescue the GL12 shRNA effects in 5Fu response, tumor sphere formation and side population changes, suggesting that ABCG2 is an important mediator for GLI2-associated 5Fu resistance. The relevance of our studies to gastric cancer patient care is reflected by our discovery that high GLI1/GLI2/ABCG2 expression is associated with a high incidence of cancer relapse in two cohorts of gastric cancer patients who underwent chemotherapy(containing 5Fu). Taken together,we have identified a molecular mechanism by which gastric cancer cells gain 5Fu resistance.     

Development of strain-specific PCR primers based on a DNA probe Fu12 for the identification of fusobacterium nucleatum subsp. nucleatum ATCC 25586T

The objective of this study was to assess the strain-specificity of a DNA probe, Fu12, for Fusobacterium nucleatum subsp. nucleatum ATCC 25586T (F. nucleatum ATCC 25586T), and to develop sets of strain-specific polymerase chain reaction (PCR) primers. Strain-specificity was tested against 16 strains of F. nucleatum and 3 strains of distinct Fusobacterium species. Southern blot hybridization revealed that the Fu12 reacted exclusively with the HindIII-digested genomic DNA of F. nucleatum ATCC 25586T. The results of PCR revealed that three pairs of PCR primers, based on the nucleotide sequence of Fu12, generated the strain-specific amplicons from F. nucleatum ATCC 25586T. These results suggest that the DNA probe Fu12 and the three pairs of PCR primers could be useful in the identification of F. nucleatum ATCC 25586T, especially with regard to the determination of the authenticity of the strain.