The Relationships among Transmission Frequency, Male Recombination and Progeny Production in DROSOPHILA MELANOGASTER

The T-007 second chromosome line, which was originally isolated in 1970 from a natural population of Drosophila melanogaster at Harlingen, south Texas, has previously been shown to be associated with several unusual genetic phenomena. In the present study, two characteristics, distorted transmission frequency and male recombination, were analyzed in relation to the progeny production of T-007 heterozygous individuals. The following points were established: (1) Distorted transmission frequency in the T-007 heterozygous male was mainly due to "elimination" of T-007 chromosomes among the progeny, while no such elimination occurred for the normal partner chromosome. (2) Transmission frequency and progeny production of the T-007 heterozygous females were normal, or at least almost normal. (3) The frequency of male recombination increased with an increasing degree of distortion. This was due to an increased number of recombinants produced per male and to a decreased number of progeny receiving the T-007 chromosome.     

Parameters of Male and Female Recombination Influenced by the T-007 Second Chromosome in DROSOPHILA MELANOGASTER

The T-007 second chromosome line of Drosophila melanogaster, previously shown to contain genetic elements responsible for male recombination induction, appears to affect several parameters of recombination in females. In T-007 heterozygous females, the distribution of recombination (but not the total frequency) is changed from that observed in control females; relative increases are observed in the more proximal regions of the second, third and X chromosomes, while relative decreases are observed more distally. These changes are paralleled by altered coefficient of coincidence values and in an increased nondisjunction frequency of second chromosomes. The distribution of recombination in females is strikingly similar to that observed in males as measured along the second and third chromosomes, and the frequency of nondisjunction of the X and Y chromosomes is increased in T-007 heterozygous males. Based upon these results and responses to the effect of structurally rearranged heterologues (the "interchromosomal effect"), it is suggested that T-007 affects the preconditions for meiotic exchange in females. It is not yet known if elements responsible for these effects are the same elements responsible for the numerous other traits associated with the T-007 second chromosome.     

Developmental Stages of Genome Elimination Resulting in Transmission Ratio Distortion of the T-007 Male Recombination (MR) Chromosome of DROSOPHILA MELANOGASTER

T-007 is a Male Recombination (MR) second chromosome that induces transmission ratio distortion (at its own expense) when heterozygous with many laboratory marker chromosomes. The developmental timing of elimination of T-007 chromosomes has been investigated. About 21% of the T-007 chromosomes expected to be recovered among the progeny of heterozygous T-007 males are lost at some point between fertilization and eclosion (representing 29% of the total distortion observed in young males). Another 52% of the expected number of T-007 chromosomes are lost as a result of spermatid abortion during spermiogenesis (representing 71% of the total distortion). Abnormalities in both the number of spermatids per bundle and the structure of spermatid tails are seen at the earliest stages of spermiogenesis in T-007 males.     

Evidence for Newly Induced Genetic Activity Responsible for Male Recombination Induction in DROSOPHILA MELANOGASTER

The T-007 second chromosomal line of Drosophila melanogaster, previously shown to contain a major element, Mr, responsible for male recombination induction, also contains the genetic capability to induce male recombination activity into (nonhomologous) third chromosomes. This newly induced male recombination activity maps to the centromeric region of two third-chromosome lines that were subjected to mapping experiments. The ability of these third chromosome lines to induce male recombination accounts for previous observations concerning the ability of Mr⁺ genotypes (derived from Mr/Mr⁺ heterozygous females) to induce male recombination for only a few generations, when only second chromosomes were selected and backcrossed. The occurrence of this effect, and a similar effect induced in the homologue of T-007, suggests a possible explanation of how natural populations of D. melanogaster have come to contain such high frequencies of these "male recombination" second and third chromosomes, despite their numerous deleterious effects.     

Elements Causing Male Crossing over in DROSOPHILA MELANOGASTER

A second chromosome line of Drosophila melanogaster (Symbol: T-007) has previously been shown to be responsible for the induction of male recombination. In the present investigation, the genetic elements responsible for this phenomenon have been partially identified and mapped. A major element (Symbol: Mr, for Male recombination) locates on the second chromosome between the pr (2L-54.4) and c (2R-75.5) loci and is responsible for the large majority of male recombination. In addition, there appear to be "secondary elements" present which have the ability to induce male recombination in much reduced frequencies and which are diluted out through successive backcross generations when Mr is removed by recombination. The possible nature of these "secondary elements" is discussed.     

Hybrid dysgenesis in Drosophila: The mechanism of T-007-induced male recombination

Summary The term hybrid dysgenesis describes a syndrome of genetic effects which sometimes results when Drosophila melanogaster from wild populations are outcrossed; this syndrome often includes male recombination as well as enhanced rates of genic and chromosomal mutation, sterility, and transmission ratio distortion. In this study, we have examined the mechanism of T-007-induced male recombination by genetically characterizing third chromosomes generated by an exchange in a well-marked euchromatic region. Most recombinant chromosomes were sequentially normal, and no recessive lethal events at the point of exchange were recovered. The results demonstrate that although some recombinants may be generated by nonhomologous chromosome (or chromatid) breakage and reunion, the predominant effect of T-007 is through an enhanced rate of normal mitotic exchange. The rate of mitotic exchange is also increased by ionizing radiation and chemical mutagens; we suggest that the common factor in all three cases is the induction of single strand breaks.     

Correlation between the species radiosensitivity of animals and concentration of AT-rich sequences in DNA

The authors have revealed a positive and statistically significant correlation between the sum of T-"pure" and T-rich pyrimidine DNA clusters and radiosensitivity of animals of different species. It was demonstrated that the share of a DNA fraction rich in AT-pairs and denaturing within the temperature range from 55 to 75 degrees increases with increasing specific radiosensitivity of animals.     

Cytotoxicity of T-2 toxin and its metabolites determined with the neutral red cell viability assay.

The neutral red (NR) cell viability assay was used with various cell types of human origin to quantitate the potency of T-2 mycotoxin and its metabolites. The human melanoma SK-Mel/27 cell line was the most sensitive, with a midpoint cytotoxicity value of 2.8 ng of T-2 per ml. With the human hepatoma cell line, HepG2, the sequence of potency for a series of mycotoxins was T-2 greater than HT-2 greater than T-2 triol greater than T-2 tetraol.     

Gametic Frequency of Second Chromosomes of the T-007 Type in a Natural Population of DROSOPHILA MELANOGASTER in Texas

The T-007 second chromosome, which was isolated from a natural population of Drosophila melanogaster in south Texas in 1970, is known to show, when made heterozygous in males with a standard cn bw second chromosome, a transmission frequency (k) of 0.35—much lower than the theoretically expected 0.5. Natural populations of this species in Texas contain second chromosomes that, against the standard cn bw genetic background, are associated with distorted transmission frequencies comparable to that of the T-007 chromosome. In order to explain how such chromosomes can persist in natural populations in nontrivial frequencies, it has been postulated that, although such chromosomes show reduced k values when tested under the genetic background of a laboratory stock such as cn bw, they may show, on the average, k values larger than 0.5 under natural genetic backgrounds. If this were true, the frequency of chromosomes of the T-007 type (T chromosomes) should be higher in male than in female gametes under natural genetic backgrounds. The present study was conducted to examine this possibility. The results clearly showed that the frequency of such chromosomes was much higher among male than among female gametes, and that the transmission frequency of this type of chromosome was higher than 0.5 under natural genetic backgrounds. These results suggest that T chromosomes behave like Segregation Distorter (SD) chromosomes in natural populations of this species in Texas. A possible relationship between T-007 and SD chromosomes is suggested.     

Recombination in Drosophila Melanogaster Male

T-007 strain of Drosophila melanogaster is known to show recombination in males. The present study established the following points: (1) Clustering occurrence of recombinant, unequal recovery of complementary products of recombination, relatively high frequency of recombination around centromeric region, and relatively frequent occurrence of mosaic phenontype flies-all of these seem to indicate that a considerable fraction of male recombination in the T-007 strain is of premeiotic, or somatic origin, although a fraction still could be of meiotic origin; (2) Male recombination occurs in the third as well as in the second chromosomes, and the frequencies of recombinations are comparable between these two chromosome pairs.     

Effects of boron deficiency in cell suspension cultures of Populus alba L.

Cell suspension cultures of Populus alba L. (original cells) require at least 10 M boron for appropriate growth. Using original cells we established a cell line, T-5B, which can grow in a medium containing low levels of boron (5 M). The level of boron localized in the cell walls of T-5B cells was one-half that found in the cell walls of original cells maintained in medium containing 100 M boron, and the level of the rhamnogalacturonan II dimer, cross-linked by a borate ester, also decreased in the former. The sugar composition of whole cell walls of the T-5B cell line was similar that of the original cells, however pectic polysaccharides composed of arabinose or galacturonic acid were easily extracted from T-5B cell walls with 50 mM trans-1,2-cyclohexanediamine-N,N,N,N-tetraacetic acid. Our results suggest that boron deficiency causes a weakening of the interaction among pectic polysaccharides due to a decrease in boron-rhamnogalacturonanII cross-linkage.     

Effect of embryonic mouse cells BALB/c-3T3 on the proliferation of the human mammary cancer cell line T-47D

In the present study, we explore the effect of the cellular extracts and culture medium of the embryonic mouse cell line BALB/c-3T3 (clone A31) on the proliferation and DNA content of the human T-47D breast cancer cell line. These effects were also studied in the presence of the potent anti-estrogen ICI 164,384. All experiments were prepared in MEM medium containing 5% fetal calf serum treated with dextran charcoal, as well as the homogenization of the BALB/c-3T3 cells to obtain the cellular extract. Aliquots of cellular extracts (2%) corresponding to 2 × 10⁶ cells, or culture medium (16%), are incubated with the T-47D cells. After 9 days of culture, cellular extracts and culture medium provoke an intense proliferative effect corresponding respectively to 2 and 5 times the control value of T-47D cells. These effects on cell proliferation are correlated with DNA content. Although the anti-estrogen ICI 164,384 (5 × 10⁻⁸ M) alone decreases the proliferation of T-47D cells by half, the presence of the culture medium from the BALB/c-3T3 cells abolishes this effect and, on the contrary, increases the cell proliferation 4-fold.

It is concluded that mouse embryonic cells (BALB/c-3T3) contain factor(s) which stimulate very intensively the proliferation of hormone-dependent T-47D mammary cancer cells. This factor(s) is present in both the cell and the culture medium and can antagonize the anti-proliferative effect of the anti-estrogen ICI 164,384.     

Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain.

We have purified L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) from Gluconobacter oxydans T-100 that showed an ability to convert D-sorbitol to 2-keto-L-gulonate (2-KLGA). A genomic library of Gluconobacter oxydans T-100 was screened with a probe, a 180-bp PCR product which was obtained from degenerate oligodeoxyribonucleotides based on the elucidated sequence of the purified SDH (used as primers) and the genomic DNA of G. oxydans T-100 (used as a template). From sequencing of the DNA from a clone positive to the probe, the SNDH and the SDH were estimated to be coded in sequential open reading frames with 1,497 and 1,599 nucleotides, respectively, which was confirmed by expression of the DNA in Escherichia coli that showed both enzymatic activities. The DNA was introduced to a shuttle vector which was prepared from a plasmid of G. oxydans T-100 and pHSG298 to obtain an expression vector designated pSDH155. The production of 2-KLGA by pSDH155 in G. oxydans G624, an L-sorbose-accumulating strain, was improved to 230% compared to that of G. oxydans T-100. Chemical mutation of the host strain to suppress the L-idonate pathway and replacement of the original promoter with that of E. coli tufB resulted in improving the production of 2-KLGA. Consequently, high-level production from D-sorbitol to 2-KLGA (130 mg/ml) was achieved by simple fermentation of the recombinant Gluconobacter.     

Characterization of simian virus 40 transformed African green monkey cells (CV-1). I. Defective virion and viral genome.

Several clones of SV40 transformed CV-1 cells have been characterized for the production of T- and V-antigens and for the state of viral genome. The transformed CV-1 cells failed to produce infectious virions as assayed after sonication or cocultivation and fusion with normal CV-1 cells, and were resistant to super-infection by SV40. Some clones of the transformed cells contained V-antigens. The population of V-antigen positive cells varied from 0 to 100% depending on the passage number while the T-antigen positive cells were always 100%. The virions isolated from the transformed cells were similar in morphology to complete SV40, but lighter in density than complete SV40. In one clone, a small amount of SV40 DNA was detectable in a free state while a large proportion of the DNA hybridizable with SV40 3H cRNA was linearly integrated into the cell DNA. The free SV40 DNA was noninfectious, closed circular DNA with a size smaller than infectious SV40 DNA component I. Since the cell extracts of the transformed cells contained an agent(s) which induced T- and V-antigens in normal CV-1 cells, it was suggested that the SV40 transformed CV-1 cells contained free as well as integrated defective SV40 genomes responsible for the synthesis of T- and V-antigens.     

Repopulation of the mouse thymus after sublethal fission neutron irradiation. I. Sequential appearance of thymocyte subpopulations

The T cell composition of the thymus of sublethal fission neutron-irradiated CBA/H mice was analyzed with cytofluorometry and immunohistology, using monoclonal antibodies directed to the cell surface antigens Thy-1, T-200, MT-4, Lyt-1, Lyt-2, and MEL-14. The results of this investigation show that whole body irradiation with 2.5 Gy fission neutrons results in a severe reduction and degeneration of the cortex, whereas the medulla is affected to a lesser extent. Irradiation selects, within 24 hr, for a population of dull Thy-1+, bright T-200+, bright Lyt-1+ cells localized in the medulla. Phenotype analysis of the regeneration of the thymus, which starts at about 5 days after irradiation, reveals the sequential appearance of: 1) "null" cells, i.e., lymphoblasts negative for all tested antigens, mainly in the subcapsular area but also in the medulla; 2) Thy-1+ "only" and T-200+ "only" cells in the subcapsular area; 3) Thy-1+, T-200+ cells; and 4) Thy-1+, T-200+, MT-4+, Lyt+ cells in the cortex. In addition, an increased MEL-14 expression is observed in correlation with the expression of Thy-1 and T-200 determinants during the regeneration of the thymus. From day 10 on up to at least 150 days after irradiation, no differences can be observed in the thymus of irradiated and age-matched sham-irradiated control mice, as measured by the expression and distribution of Thy-1, T-200, MT-4, Lyt-1, Lyt-2, and MEL-14 antigens. The observed sequence in phenotype shift in the regeneration of the thymus after irradiation is discussed in view of recently published data on the differentiation of the T cell system.     

Relationship between folding and function in a sequence-specific miniature DNA-binding protein

Previously, we have described a miniature protein-based approach to the design of molecules that bind DNA or protein surfaces with high affinity and specificity. In this approach, the small, well-folded protein avian pancreatic polypeptide acts as a scaffold to present and stabilize an alpha-helical or PPII-helical recognition epitope. The first miniature protein designed in this way, a molecule called p007, presents the alpha-helical recognition epitope found on the bZIP protein GCN4 and binds DNA with nanomolar affinity and exceptional specificity. In this work we use alanine-scanning mutagenesis to explore the contributions of 29 p007 residues to DNA affinity, specificity, and secondary structure. Virtually every residue within the p007 alpha-helix, and most residues within the p007 PPII helix, contribute to both DNA affinity and specificity. These residues include those introduced to make specific and nonspecific DNA contacts, as well as those that complete the miniature protein core. Moreover, there exists a direct correlation between the affinity of a p007 variant for specific DNA and the ability of that variant to select for specific DNA over nonspecific DNA. Although we observe no correlation between alpha-helicity and affinity, we observe a limited correlation between alpha-helicity and sequence specificity that emphasizes the role of coupled binding/folding in the function of p007. Our results imply that formation of a highly evolved set of protein.DNA contacts in the context of a well-packed hydrophobic core, and not the extent of intrinsic alpha-helical structure, is the primary determinant of p007 function.     

Monoclonal anti-idiotype induces protection against the cytotoxicity of the trichothecene mycotoxin T-2

An IgG1 mAb, designated HD11, specific for the trichothecene mycotoxin T-2 and capable of neutralizing its cytotoxicity was used to generate a syngeneic monoclonal anti-Id antibody. The generated anti-Id mAb, designated DE8, specifically bound to HD11 anti-T-2 mAb, and not to IgG1 mAb of irrelevant specificity or to normal mouse Ig. DE8 inhibited the binding of HD11 anti-T-2 to T-2-BSA-coated plates, whereas a control anti-Id mAb did not, suggesting recognition of an Id determinant associated with the T-2 binding site of HD11. Moreover, the binding of HD11 to DE8 and that of DE8 to HD11 were specifically inhibited by free T-2 mycotoxin. DE8 mAb was efficient in abrogating the protective effect of HD11 in the cytotoxicity of T-2 on the human epidermoid carcinoma cell line Hep-2. In vivo immunization of BALB/c mice with DE8 conjugated to KLH induced an anti-T-2 antibody titer comparable to that obtained with T-2-OVA immunization, whereas immunization with unconjugated DE8 resulted in a lower titered anti-T-2 response. Immunization with DE8-keyhole limpet or with unconjugated DE8 induced anti-T-2 antibody responses characterized by expression of "HD11-like" Id and by protection against T-2 cytotoxicity. However, the T-2-OVA-induced anti-T-2 response lacked the HD11+ Id and was only partially protective against T-2 cytotoxicity. This represents the first demonstration of the use of an anti-Id based vaccine in the in vivo induction of a protective antibody response against the cytotoxicity of a nonproteinaceous, small m.w. biologic toxin, whose very toxic nature precludes its use as the immunogen.     

Changes of enzymes and factors involved in DNA synthesis during wheat embryo germination

We have previously purified and characterized wheat germ DNA polymerases A and B. To determine the role played by DNA polymerases A and B in DNA replication, we have measured the level of their activities during wheat embryo germination. The level of cellular proteins known to be associated with DNA synthesis such as PCNA and DNA primase were also investigated. The activity of DNA polymerase A gradually increased reaching a maximal level at 12 h after germination. Three days later, only a residual activity was detected. DNA polymerase B showed the same pattern during germination with very similar changes in activity. Our results indicate a striking correlation between maximal activities of DNA polymerase A, DNA polymerase B and optimal levels of DNA synthesis. These results support a replicative role of these enzymes. The activity of wheat DNA primase that copurifies with DNA polymerase A also increases during wheat germination. Taking together all its properties, and in spite of its behaviour with some inhibitors, DNA polymerase A may be considered as the plant counterpart of animal DNA polymerase . Concerning DNA polymerase B we have previously shown that PCNA stimulates its processivity. Besides studying the changes of DNA polymerases A and B and DNA primase we have also studied changes in PCNA during germination. We show that PCNA is present in wheat embryos at a constant relatively high level during the first 24 h of germination. After 48 h, the absence of PCNA is concomitant with an important decrease in DNA polymerase B activity. In this report we confirm the behaviour of DNA polymerase B as a -like activity.Département de Biologie, Université de Drah-Lmraz,Fez, Maroc     

离子束介导甘蓝全DNA转化拟南芥菜的分子分析

30 keV的Ar+离子束在1.5×1017 ions/cm2的注入剂量下介导外源甘蓝全DNA导入模式植物拟南芥菜,在94株转化当代植株中,有6株表型产生变异。以其中的一株(T-5)作为研究对象,用80条10碱基随机引物对该株和其子代变异株基因组作随机扩增的多态性DNA分析,引物S176 在T-5和其变异子代T-5-2中扩增出了相同分子量的变异新条带T-5S176-620。T-5S176-620的碱基序列和拟南芥菜基因组序列进行同源比对,结果表明该片段不属于拟南芥菜基因组,Southern杂交实验证明该片段来自供体甘蓝基因组。但是,根据T-5S176-620序列设计的引物不能从甘蓝基因组中扩增出预期长度的DNA片段,结合离子束介导外源全DNA转化的特点和过程,探讨了其中可能的机制。     

Cytotoxicity of T-2 toxin and its metabolites determined with the neutral red cell viability assay

The neutral red (NR) cell viability assay was used with various cell types of human origin to quantitate the potency of T-2 mycotoxin and its metabolites. The human melanoma SK-Mel/27 cell line was the most sensitive, with a midpoint cytotoxicity value of 2.8 ng of T-2 per ml. With the human hepatoma cell line, HepG2, the sequence of potency for a series of mycotoxins was T-2 greater than HT-2 greater than T-2 triol greater than T-2 tetraol.