Population genetics theory of concerted evolution and its application to the immunoglobulin V gene tree

Summary The previous simple model for treating concerted evolution of multigene families has been revised to be compatible with various new observations on the immunoglobulin variable region family and other families. In the previous model, gene conversion and unequal crossing-over were considered, and it was assumed that genes are randomly arranged on the chromosome; neither subdivision nor correlation of gene identity and chromosomal distance were considered. Although this model satisfactorily explains the observed amino acid diversity within and between species, it fails to predict the very ancient branching of the mouse immunoglobulin heavy chain V-gene family. By incorporating subdivided structure and genetic correlation with chromosomal distance into the simple model, the data of divergence may be satisfactorily explained, as well as the rate of nucleotide substitution and the amino acid diversity. The rate at which a V-gene is duplicated or deleted by conversion or by unequal crossing-over is estimated by the new model to be on the order of 10^–6 per year. The model may be applicable to other multigene families, such as those coding for silkmoth chorion or mammalian kallikrein.Contribution no. 1560 from the National Institute of Genetics, Mishima, 411 Japan     

Does Tn10 transpose via the cointegrate molecule?

Summary It has been well established that Tn3 and its relatives transpose from one replicon to another by two successive reactions: formation of the cointegrate molecule and resolution from it. Whether or not the 9300 base pair tetracycline resistance transposon Tn10 transposes in the same manner as Tn3 was investigated by two methods.In the first method, 55, a lambda phage carrying Tn10 was lysogenized in an Escherichia coli strain carrying a Tn10 insertion; the phage has a deletion in attP, hence it was lysogenized in a Tn10 sequence in the E. coli chromosome by reciprocal recombination. The chromosomal structure in these lysogens is equivalent to the Tn10-mediated cointegrate molecule of lambda and the E. coli chromosomal DNA. The stability of the cointegrate molecule was examined by measuring the rate of excision of lambda from the host chromosome, and was found to be stable, especially in a Rec^- strain. Because of this stability, the cointegrate molecule should be accumulated if Tn10 transposes via the cointegrate molecule. Then, we examined the configuration of products made by transposition of Tn10 from 55 to the E. coli chromosome. The cointegrate molecule was found in products of Tn10 transposition in a Rec⁺ strain at a frequency of 5% per Tn10 transposition, but this molecule could not be found in a Rec^- strain. Since transposition of Tn10 was recA-independent, absence of the cointegrate molecule formed in a RecA^- strain strongly suggested that the cointegrate molecule is not an obligatory intermediate of transposition of Tn10.In the second method, mobilization of pACYC177 by R388 and by R388:: Tn10 was examined. The pACYC177 plasmid was mobilized by R388::Tn10 at a frequency of 10^-4 per donor but not by R388. It occurred, in most cases, by inverse transposition of R388::Tn10 to pACYC177 forming plasmids such as pACYC177::IS10-R388-IS10. Mobilization of pACYC177 by a Tn10-mediated cointegrate in the form of pACYC177::Tn10-R388-Tn10 was not observed in crosses using a Rec^- donor. These observations also suggested that transposition of Tn10 in Rec^- cells does not occur via the cointegrate molecule.     

Characterization of newly isolated monoclonal antibodies against MHC of a Japanese wild mouse

We have already developed nine B10.MOL congenic strains carrying H-2 haplotypes derived from Japanese wild mice, Mus musculus molossinus, with the C57BL/10 genetic background. To obtain monoclonal antibodies against the H-2 antigen of the Japanese wild mouse, we carried out cell fusion using spleen cells from the animal immunized with one of the B10.MOL strains, B10.MOL-SGR (H-2 ^wm7). As a result, 19 hybridomas producing monoclonal antibodies were produced. Analysis with the intro-H-2 recombinants derived from B10.MOL-SGR indicated that 8 of them reacted with the class I and II with the class II molecule. The class I antibodies were tested for their cross -reactivities on wild mice and on the panels of standard inbred and B10.MOL strains. Most of the antibodies reacted with both the Japanese wild mice and the other subspecies, including standard inbred, while two antibodies highly specific for the donor H-2K region reacted with only three wild-derived mice, two M. m. molossinus from Anj o and Shizuoka, Japan, and one M. m. domesticus from Pigeon, Canada. In addition, all of the other four antibodies reactive with the K antigen of B10.MOL-SGR also reacted with the same three wild mice. The wild mice belonging to different subspecies might share very similar H-2K antigenic determinants in spite of their genetic and geographical remoteness.     

Effects of colcemid and taxol on microtubules and intermediate filaments in chick embryo fibroblasts

Reports on how changes in microtubule (MT) distribution or polymerization affect the distribution of intermediate filaments (IFs) differ. Therefore, we have used cytoimmunofluorescence techniques and electron microscopy to systematically examine and compare the arrangements of MTs and IFs in cultures of chick embryo fibroblasts under the following conditions: at different times during the cell cycle, in the presence of Colcemid or of taxol, in the presence of both drugs in succession or simultaneously in varying ratios, and during recovery from treatment with Colcemid or taxol. We have found that depolymerization of MTs by 1 microM Colcemid resulted in the rapid formation of massive IF-cables, structures distinct from "collapsed IFs" or "juxtanuclear coils." Neither the rapid formation of IF-cables nor their dispersion during recovery required protein synthesis. Cells treated with 10 microM taxol rapidly formed MT-bundles, as well as aggregates of intertwining IFs, termed "IF-skeins." MT-bundles and IF-skeins displayed strikingly complementary distributions. This reciprocal distribution of packed MTs and IFs was also obvious in untreated anaphase and telophase cells. When 10 microM taxol and 1 microM Colcemid were applied simultaneously, the complementary distributions of MT-bundles and IF-skeins mimicked those in taxol alone. This ability of taxol to block Colcemid's effects was concentration dependent. Decreasing the taxol: Colcemid ratio allowed the depolymerization of MTs, which correlated with the formation of IF-cables.     

On the constancy of the evolutionary rate of cistrons

Summary The variations of evolutionary rates in hemoglobins and cytochrome c among various lines of vertebrates are analysed by estimating the variance. The observed variances appear to be larger than expected purely by chance.If the amino acid substitutions in evolution are the result of random fixation of selectively neutral or nearly neutral mutations, the evolutionary rate of cistrons can be represented by the integral of the product of mutation rate and fixation probability in terms of selective values around the neutral point. This integral is called the effective neutral mutation rate.The influence of effective population number and generation time on the effective neutral mutation rate is discussed. It is concluded that the uniformity of the rate of amino acid substitutions over diverse lines is compatible with random fixation of neutral or very slightly deleterious mutations which have some chance of being selected against during the course of substitution. On the other hand, definitely advantageous mutations will introduce significant variation in the substitution rate among lines. Approximately 10% of the amino acid substitutions of average cistrons might be adaptive and create slight but significant variations in evolutionary rate among vertebrate lines, although the uniformity of evolutionary rate is still valid as a first approximation.Contribution No. 813 from the National Institute of Genetics, Mishima, Shizuokaken 411 Japan. Aided in part by a grant-in-aid from the Ministry of Education, Japan.     

Amino acid uptake in the peripheral nerve of the rat

When rat sciatic nerves were incubated with C¹⁴l-lysine, l- or d-glutamate, or d-l γ-aminoisobutyrate, the labeled compounds penetrated the nerve, and the level of lysine and leucine after 1 hr was higher in the nerve than in the medium. The level increased with time, and at 24 hr glutamate levels also were higher in the nerve than in the medium. Lowering the temperature strongly inhibited uptake, while other conditions such as absence of glucose, absence of sodium, or the presence of cyanide inhibited uptake by nerve less than uptake by brain slices. The uptake against a concentration gradient, and inhibitions of this uptake by metabolic inhibitors and by structural analogs, were interpreted as showing the presence of transport processes for amino acids in peripheral nerves with characteristics similar to such transport processes in the central nervous system.     

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.