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Two loci (the incompatibility or mating type loci), a and b , govern the life cycle of Ustilago maydis , a fungal pathogen of corn. U. maydis diploids heterozygous at both a and b ( a [unk] b [unk]) form mycelial colonies (Fuz + phenotype), induce tumors, and produce teliospores able to undergo meiosis. We report here the isolation and characterization of nonmycelial (Fuz - ) derivatives. These Fuz - diploids have allowed us to examine the requirement of a [unk] and b [unk] in maintenance of filamentous growth and tumor-inducing ability. The Fuz - diploids are of four classes: two are inferred to be homozygous for b ( a [unk] b =); the other two are shown to be homozygous for a ( a = b [unk]). These observations confirm the requirement for b [unk] and reveal the requirement for a [unk] in filamentous growth. U. maydis is thus like other basidiomycetes that have two loci governing growth of the dikaryotic filament. The mating behavior of the Fuz - diploids indicates that heterozygosity at a or b does not block mating. Although both a = b [unk] and a [unk] b = diploids are nonmycelial, they differ in that a [unk] b = diploids are nonpathogenic, whereas a = b [unk] diploids are pathogenic and produce teliospores able to undergo meiosis. These findings substantiate previous, more limited observations. They demonstrate that ability to induce tumors and produce normal teliospores requires different alleles at b but not at a .

The synthesis of the A-gliadin protein fraction derived from the endosperm of the grain of hexaploid bread wheats ( Triticum aestivum L.), which is toxic in celiac disease, was associated with the α arm of the 6A chromosome through use of the substitution lines of “Cheyenne” chromosomes in “Chinese Spring”. The association was made through the use of ditelocentric stocks of Chinese Spring. The synthesis of many other gliadin components in the gel electrophoretic patterns of these two varieties could be associated with particular chromosomes as well. All genes detected were located in the chromosomes of homoeologous groups 1 and 6. It is possible to remove some of the proteins toxic to people with celiac disease from wheat (flour) by chromosome manipulation. If the toxic factor is not widely distributed among the storage protein components, it may be possible to produce a wheat that would be safe for celiac patients to eat.

There is increasing evidence that mouse cells of different strains contain different numbers and biologic classes of naturally-integrated type-C RNA viruses. The present studies have examined the cellular regulation of one class of virus, xenotropic in host range, which is common to mice of NIH Swiss and NZB strains. This virus is spontaneously released at high level by NZB embryo cells but its production by NIH Swiss embryo cells is below detectable levels. Quantitative analysis of the chronic level of virus release by embryo cell lines established in tissue culture from genetic crosses of NIH and NZB strains has demonstrated the existence of a partially dominant gene influencing virus release. Evidence is presented indicating that this gene may act to provide a factor necessary for expression of a specific class of endogenous virus.

Different strains of the established human cell line HeLa differ substantially in sensitivity to ethyl methanesulfonate (EtMes). The EtMes doses effective for either cytotoxicity or mutation induction in a line of HeLa S3 cells are about 1/10th those required in the CCL2 HeLa line of the American Type Culture Collection. By plating the sensitive HeLa S3 line in the presence of highly cytotoxic doses of EtMes, we obtained a clone (designated A6) that displays about 7-fold greater resistance to EtMes toxicity. This A6 isolate is also cross resistant to other simple monofunctional alkylating agents—exhibiting about 4-fold increased resistance to methyl methanesulfonate and 10- to 15-fold increased resistance to N -methyl- N ′-nitro- N -nitrosoguanidine but is similar to the S3 parent in sensitivity to mitomycin C, UV radiation, and γ-rays. In contrast to the results for cytotoxicity, the A6 variant and the S3 parent showed the same high susceptibility to EtMes induction of ouabain-resistant mutations. This is direct biological evidence that different alkylation lesions are normally responsible for mutagenic and cytotoxic effects. The S3 and A6 cell lines may differ in DNA repair capability specific to certain potentially lethal alkylation products. The comparative sensitivity of the A6 cells to alkylation mutagenesis may also prove useful in cell genetic studies by facilitating the generation of multiple mutants for recessive alleles and permitting exceptionally sensitive detection of specific mutagenic effects.

Replication patterns of four classes of heterochromatin in the chromosomes of maize were analyzed. Centromeric heterochromatin, distal heterochromatin of the B chromosomes, and knob heterochromatin all have asynchronous late replication, knob heterochromatin being the last chromosomal DNA to complete replication. Nucleolus organizer heterochromatin replicates during the period of euchromatin DNA replication. The proportion of the S period involved in asynchronous late replication is directly dependent on the amount of heterochromatin in the nucleus. Both additional knob and B-chromosome heterochromatin participate in this effect. Each additional B chromosome produces an increment in the proportion of nuclei with asynchronous replication; in nuclei with 12 B chromosomes, the proportion of the S phase showing asynchronous late replication is more than 3 times greater than that in nuclei without B chromosomes. The data are consistent with the hypothesis that delayed DNA replication of knob heterochromatin is responsible, in some genotypes, for the loss of chromosome segments and for B-chromosome nondisjunction in the second pollen grain mitosis.

Hybridization studies with a [ 3 H]DNA probe from an endogenous baboon type C virus were used to detect nucleic acid sequences of type C virus in primate cellular DNA. Sequences related to those of the baboon type C virus are found in all other Old World monkey species, higher apes, and man.

Data are presented that indicate that the z locus of Drosophila melanogaster represses w locus activity, but the repression is effective only on paired or physically adjacent w loci. Various mutant alleles of z and w were combined in a series of different doses to determine the effect of dosage and physical position in the nucleus on gene expression. In z/z individuals, paired white alleles fail to be expressed, while unpaired alleles are expressed normally. The results are discussed in terms of a model postulating that the z gene product represses the transcription of w + by complexing with an RNA produced by part of the white locus itself. In order to be effective in repression, there must be two w + genes producing the RNA in a limited volume in the nucleus. Such a model necessarily imposes a specific architecture on the chromatin of interphase nuclei.

The inheritance of six polymorphic loci mapping in the rRNA-encoding (rDNA) region of the inverted repeat sequence of chloroplast DNA (cpDNA) was scored in hybrid subclones derived from reciprocal interspecific crosses between the green algae Chlamydomonas eugametos and Chlamydomonas moewusii . In order to enhance the detection of cells that had undergone recombination between parental cpDNAs, hybrids were selected that inherited a chloroplast antibiotic-resistance marker contributed by the mating-type-minus( mt - ) parent, the parent that normally contributes fewer cpDNA molecules. The major findings of this study can be summarized as follows. ( i ) The majority of the hybrids (14/17) were recombinant for cpDNA markers in the 10-kilobase-pair rDNA region under study. ( ii ) Only one allele of each polymorphic cpDNA locus was ever detected in the hybrids, thus suggesting that newly recombined rDNA sequences in one copy of the inverted repeat are rapidly spread to the other by a copy-correction mechanism. ( iii ) Chloroplast streptomycin-resistance ( sr-2 ) and erythromycin-resistance ( er-nM1 ) loci, although showing little or no genetic linkage, were mapped to the 16S and 23S rRNA gene regions of the cpDNA, respectively, by virtue of their perfect coinheritance with polymorphic markers within these genes. ( iv ) cpDNA markers associated with a putative intron of the C. eugametos 23S rRNA gene were inherited by all 17 hybrids. Such a result is similar to that observed for certain alleles of the large rRNA gene of yeast mitochondria in crosses between ω + and ω - strains.

The human alpha-globin and phosphoglycollate phosphatase (EC 3.1.3.18) genes have been regionally localized to the short arm of human chromosome 16 (HC16). This was accomplished by fusing mouse fibroblasts (A9) to human fibroblasts that contain a reciprocal translocation between the long arms of chromosomes 16 and 11. The murine A9 cells are deficient in adenine phosphoribosyltransferase (APRT), an enzyme present on the long arm of HC16 (HC16q). Hybrid cells were grown in selection culture medium that required the cells to retain human APRT. Therefore, the hybrids exhibited stable retention of the entire HC16 or the rearranged chromosome containing HC16q. We isolated five independent primary and secondary hybrid cell lines which retained either HC16 or HC16q at a high frequency. The presence of human alpha-globin genes in the various clones was established directly by DNA extraction and hybridization to a cDNA probe for human alpha-globin genes. Autoradiographs showed that hybrid cells containing the long arm, but not the short arm, of HC16 showed only the background mouse bands. Hybrid cells that retained the entire HC16 demonstrated the band(s) containing the human alpha-globin genes. Hybrid cells that contained HC16 with its alpha-globin genes were then placed in culture medium that contained diaminopurine, which is lethal for cells containing APRT. These counter-selected hybrid cells had lost HC16 and also lost the human alpha-globin genes as determined by blot hybridization. The presence of alpha-globin gene sequences in the hybrid clones was concordant with HC16 only and not with any other human chromosome. These results confirm the assignment of alpha-globin genes to HC16 and localize the genes to the short arm. We also assign the locus for phosphoglycollate to the short arm of HC16.

Haploid yeast cells normally contain either the MAT a or MAT α mating-type allele and cannot undergo meiosis and spore formation. If both mating-type alleles are present as a consequence of chromosome III disomy ( MAT a/ MAT α), haploids initiate meiosis but do not successfully form spores, probably because the haploid chromosome complement is irregularly partitioned during meiotic nuclear division. We have demonstrated that the ochre-suppressible mutation spo13-1 enables haploid yeast cells disomic for chromosome III and heterozygous at the mating-type locus to complete meiosis and spore formation, yielding two haploid spores. Previous studies have shown that the absence of the wild-type SPO13 gene function permits diploid cells to bypass homologous chromosome segregation at meiosis I and proceed directly to meiosis II. During spo13-1 haploid meiosis, cells enter prophase of meiosis I. Genetic recombination, monitored on the chromosome III disome, occurs at levels similar to those seen in diploids, indicating that the level of exchange between homologs is an autonomous property of individual chromosomes and not dependent on exchange elsewhere in the genome. Exchange is then followed by a single meiosis II equational chromosome division. Recombination in spo13-1 haploids is blocked by the spo11-1 mutation, which also eliminates recombination between homologous chromosomes during conventional diploid meiosis. We conclude that Spo + haploids expressing both a and α mating-type information attempt a SPO13 -dependent meiosis I division, and that this division, in the absence of paired homologous chromosomes, is responsible for the failure of such haploids to complete normal gametogenesis. Our observations support the conclusion that initiation and completion of meiosis II and spore formation are not dependent on either completion of meiosis I or the presence of a diploid chromosome complement.

RNA preparations containing 70-80% mouse κ-chain mRNA have been prepared. The remainder consists of many RNA species, each of which represents a small fraction of the total RNA. The κ-chain mRNA preparation hybridizes with mouse liver DNA with bi-phasic kinetics, indicating that it consists of two fractions —“unique” and “reiterated.” Competition hybridization experiments show that the homology among the unique fractions from different mRNAs is the same as the homology among the amino acid sequences of the corresponding κ-chains. Hence, in addition to the C-region (constant-region) sequences, (most of) the V-region (variable-region) sequences are also derived from unique germ line genes. The reiterated fractions from different κ-chain mRNAs show essentially complete homology with each other. This fraction seems to consist mostly of sequences which do not code for amino-acid sequences of the secreted polypeptide chain, i.e., the “external” section of the mRNA molecule. It is concluded that the number of germ line genes is too small to account for the observed diversity of antibody molecules.

The experiments described in this paper implicate chloroplast protein synthesis in the regulation of nuclear DNA replication. The inhibition of nuclear DNA replication in the lower eukaryote, Chlamydomonas reinhardi strain 21gr , was examined after growth of cells with a series of antibiotics (streptomycin, neamine, spectinomycin, cleocin, chloramphenicol, and rifampicin) each of which has a known effect upon chloroplast RNA or protein synthesis in this organism. Each antibiotic inhibited nuclear DNA replication at drug concentrations at which there was little or no inhibition of adenine incorporation into chloroplast DNA. That chloroplast DNA was replicating under these conditions rather than merely being repaired, was shown first by the high incorporation rates and second by a 14 N- 15 N density transfer experiment in which chloroplast DNA doubled in the presence of streptomycin, while no incorporation into nuclear DNA was detected. A small DNA peak, Component III, located between nuclear and chloroplast DNA's in CsCl gradients, possibly mitochondrial, was more pronounced in DNA from antibiotic-inhibited cultures than from controls.

Dividing cells from persons with Bloom's syndrome, an autosomal recessive disorder of growth, exhibit increased numbers of chromatid breaks and rearrangements. A highly characteristic feature of the chromosome instability in this syndrome is the tendency for exchanges to occur between chromatids of homologous chromosomes at homologous sites. In the present experiments, a cytogenetic technique by which the sister chromatids of a metaphase chromosome are stained differentially has been used to demonstrate a striking and possibly specific, but hitherto unrecognized, increase in the frequency with which sister chromatids also exchange segments. The cells were grown in bromodeoxyuridine and stained with 33258 Hoechst and Giemsa. Whereas phytohemagglutinin-stimulated lymphocytes from normal controls had a mean of 6.9 sister chromatid exchanges per metaphase (range 1-14), those from persons with Bloom's syndrome had a mean of 89.0 (range 45-162). Normal frequencies of sister chromatid exchanges were found in cells heterozygous for the Bloom's syndrome gene, and also in cells either homozygous or heterozygous for the genes of the Louis-Bar (ataxia telangiectasia) syndrome and Fanconi's anemia, two other rare disorders characterized by chromosome instability. In a differentially stained chromatid interchange configuration discovered during the study, it was possible to determine the new distribution of both sister and non-sister-but-homologous chromatids that had resulted from numerous exchanges. By following shifts in the pattern of staining from chromatid to chromatid, visual evidence was obtained that the quadriradial configurations long recognized as characteristic of Bloom's syndrome represent exchanges between homologous chromosomes, apparently at homologous points. We postulate that the increase in the frequency of exchanges between nonsister-but-homologous chromatids and those between sister chromatids in Bloom's syndrome represents aspects of one and the same disturbance. A study of this phenomenon in relation to the clinical features of Bloom's syndrome may be helpful eventually in understanding the biological significance of chromatid exchange in somatic cells.

We have recently demonstrated that normal hemopoietic cells express RNA sequences that are homologous to sequences specific for the Friend erythroleukemia virus genome [Bernstein, A., Gamble, C., Penrose, D. & Mak, T. W. (1979) Proc. Natl. Acad. Sci. USA 76, 4455-4459]. In this communication, we report that the Fv-2 locus, the major genetic determinant controlling host susceptibility to erythroleukemia induction by Friend leukemia virus, also controls the expression of endogenous sequences related to the replication-defective component of Friend leukemia virus, Friend spleen focus-forming virus (SFFV), in normal uninfected mice. Two independent congeneic pairs of mice [C57BL/6 (B6) and B6.S; B6 and B6.C(H-7 b )], differing only in a small region of the mouse genome including the Fv-2 locus, were used for this purpose. In both cases, molecular hybridization analysis indicated that the presence of SFFV-related RNA sequences in normal mice was associated with the Fv-2 s allele: bone marrow or spleen cellular RNA from Fv-2 rr B6 mice contained no detectable SFFV-related sequences, whereas their congeneic Fv-2 ss pairs contained relatively high levels of these RNA sequences. The absence of these RNA sequences in Fv-2 rr mice was not due to deletion of these sequences from the DNA of Fv-2 rr mice. Repopulation of lethally irradiated Fv-2 rr mice with syngeneic Fv-2 rr bone marrow cells did not lead to any increase in the levels of these SFFV-related RNA sequences, suggesting that the expression of these sequences is still reduced or inhibited in actively cycling Fv-2 rr hemopoietic cells. Infection with Friend leukemia virus resulted in the appearance of high levels of RNA homologous to SFFV-specific sequences in the leukemic spleens of B6.S ( Fv-2 ss ) mice, whereas these cellular RNA sequences could not be detected in the spleens of Friend virus-infected B6 ( Fv-2 rr ) mice. The demonstration that the same gene locus controls both the expression of exogenous SFFV-specific sequences and erythroleukemia induction by Friend leukemia virus suggests that these sequences may be necessary for erythroleukemic transformation. In addition, the finding that the Fv-2 gene locus controls the expression of endogenous SFFV-related sequences suggests that these sequences may also be involved in normal hemopoiesis.

A temperature-sensitive lethal mutant of Drosophila melanogaster called ecd 1 becomes deficient in ecdysone, as measured by a radioimmunoassay, when there is a shift in temperature from 20° to 29° at various stages of development. Associated with the ecdysone deficiency at 29°, there are abnormalities in larval and imaginal development and the adult functions. When the shift occurs early in third-instar stage, the mutant larvae grow to full size but fail to pupariate, and instead remain living larvae for as long as 3 weeks. These larvae, which have only about 5% as much ecdysone as the wild-type at the time of pupariation, can be induced to pupariate at 29° by ecdysone in their food, indicating that the pupariation block results from an ecdysone deficiency. A shift to 29° later in the third-instar stage does not prevent pupariation of the mutant, but the imaginal discs fail to complete differentiation, although the discs can differentiate at 29° after transplantation to the normal environment of a wild-type host. A shift to 29° early in the first-instar stage blocks a subsequent rise in ecdysone titer and results in extensive developmental defects. Mutant adults become sterile at 29°, and the ecdysone titer in the females concomitantly decreases to 13% of the wild-type value. Mutant larval ovaries transplanted to wild-type female hosts continue to develop and produce competent eggs at 20°, but when the adult hosts are put at 29° the transplanted ovaries become sterile, suggesting that the ecdysone needed for female fertility is synthesized autonomously by ovarian tissue. In contrast to these effects of a shift to 29° during larval, pupal, and adult stages, there is a normal increase in ecdysone titer and normal development in mutant embryos grown at 29°. The insensitivity of the embryo to the ecd 1 mutation might be due to a maternal contribution of components needed for ecdysone synthesis during the embryonic stage.

A black 25-year-old woman and her father have a fast-moving α chain variant in an amount of 8% (the father) and 18% (the daughter). Structural data indicate that this chain has been elongated by the addition of three amino-acid residues to give the sequence: -Pro(114)-Ala(115)-Glu(116)-Phe(117)-Thr(118)- Glu-Phe-Thr- Pro(119)-Ala(120)-.The underlying genetic alteration responsible for hemoglobin Grady appears, therefore, to be a tandem duplication of nine base pairs which may have arisen by a process of mismatched intragenic crossing over. Functional and physicochemical properties of the variant are not greatly altered, and hematological data are normal.

In contrast to standard-height wheat genotypes, short-statured wheats having major genes for dwarfness do not show increased seedling growth after treatment with gibberellic acid. Endogenous gibberellic acid induces synthesis of amylase in the endosperm of germinating seeds, but the amount of amylase synthesized is greatly increased by exogenous gibberellic acid treatment in standard-height and in short-statured wheats that have dwarfing genes from the variety “Norin 10.” “D6899,” which has the “Tom Thumb” gene for height reduction, had about one-fourth of the amylase activity of standard-height and Norin 10-derived, short-statured wheats. This genotype showed little or no increased amylase activity after gibberellic acid treatment. Genetic analyses showed that the amount of amylase synthesized was controlled by a single gene and was dependent on the number of copies of the structural gene present in the endosperm. Dwarfism in wheat may be related to a blockage in gibberellic acid utilization because other workers have found that the amount of endogenous amylase synthesized in Norin 10-derived, short-statured wheats is not growth-limiting, but it is not known if low amylase synthesis is related to dwarfism in the Tom Thumb derivative. No recombinants were recovered in a small population, suggesting that the Tom Thumb gene may pleiotropically affect plant height and the lack of response to gibberellic acid in amylase synthesis and seedling growth.

Two human choriocarcinoma cell lines were shown to be deficient in adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) such that they did not produce bands on starch gels after electrophoresis and histochemical staining. Radiometric assay indicated that their ADA specific activity was approximately 2% that of HeLa (human) cell controls. Subclone analysis of one of the lines indicated that this deficiency was representative of individual cells of the line. After fusion of these cells with mouse fibroblasts having high ADA activity, most independently isolated hybrid clones expressed one of two, or both, additional (to the mouse) bands of ADA activity after electrophoresis. The expression of these extra bands in hybrids was dependent upon actual fusion. The phenomenon was observed in 30 of 45 independently derived hybrid clones from four different fusion experiments involving two different parental lines from each species. The pattern of appearance of the extra bands in independent hybrid clones and the tendency of a hybrid clone to lose one of the extra bands through subsequent passages suggests that the bands were the products of human genetic material. The extra bands electrophoretically comigrated with human ADA 1 and 2 from human ADA-1-2 heterozygotes and the faster-migrating of the two extra bands comigrated with human ADA 1 from HeLa cells. Therefore, we suggest that the bands appearing in hybrids are the products of the 1 and 2 alleles of the human ADA locus. The human cells used for fusion were deficient in ADA activity but contained the genetic information for ADA 1 and 2. Fusion with mouse cells having ADA activity resulted in the activation of both human gene products coded for on separate homologous chromosomes. We conclude that the human ADA locus is under manipulatable genetic regulation.

Two symmetrically located deletions of approximately 100 base pairs each have been identified in chloroplast DNA of Chlamydomonas reinhardtii . Although present in a mutant strain that requires acetate for growth, both deletions have been shown to be distinct from the nonphotosynthetic phenotype of this strain. These physical markers in the chloroplast genome and maternally inherited genetic markers showed strict cotransmission in reciprocal crosses. Thus, our results are consistent with the location of the well-characterized maternally inherited genetic markers in chloroplast DNA of C. reinhardtii .

DNA-coated gold particles were introduced into meristems of immature soybean seeds using electric discharge particle acceleration to produce transgenic fertile soybean plants. The lineages of integrated foreign DNA in two independently transformed plants were followed in the first (R 1 ) and second (R 2 ) generation of self-pollinated progeny. One plant (4615) was transformed with the Escherichia coli genes for β-glucuronidase and neomycin phosphotransferase II; the other (3993) was transformed only with the gene for β-glucuronidase. Segregation ratios for the introduced gene(s) were approximately 3:1 for plant 4615 and 1:1 for plant 3993 in the R 1 generation. DNA analysis showed 100% concordance between presence of the foreign gene sequences and enzyme activity. Moreover, all copies of the foreign genes are inherited as a unit in each plant. Plant 3993 segregated in a 1:1 ratio in the R 2 generation. R 1 plants derived from plant 4615, which expressed both genes, gave either 100% or 3:1 expression of both genes in the R 2 generation, demonstrating recovery of both homozygous and heterozygous R 1 plants. Our results show that foreign DNA introduced into soybean plants using electric discharge particle acceleration can be inherited in a Mendelian manner. Results also demonstrate cotransformation of tandem markers and show that both markers are inherited as closely linked genes in subsequent generations. These results indicate that whole plants can be derived from single transformed cells by a de novo organogenic pathway.

In order to clarify the mechanisms responsible for the observed linkage disequilibrium, such as that found between markers in the major histocompatibility complex of man and mouse, linkage disequilibrium between two linked loci was studied for a finite population with a subdivided structure. The infinite allele model was used. In analogy with the subdivision of the inbreeding coefficient, the linkage disequilibrium coefficient was subdivided, and various variance components of disequilibrium were defined. It was found that the disequilibrium components may get very large when migration is limited if the correlation of alleles at the two loci within a colony is taken relative to that of the entire population. In other words, with limited migration, random genetic drift of gamete types prevails in each colony. A possible test in which the variance components of disequilibrium are compared is suggested; the test discriminates between epistatic natural selection and limited migration, showing the former as the main cause of the observed linkage disequilibrium. It is pointed out that the major histocompatibility complex polymorphism is in accord with the population genetics model of multigene families that incorporate gene conversion or double unequal crossing-over between the loci in the supergene family.

The sex-limited “alba” genetic polymorphism in wing color of Colias butterflies has been studied with respect to potential selective pressures on this locus. Alba female pupae, carrying at least one dominant A allele, redirect resources, used by aa pupae for pigmentation, to other metabolic ends. Associated with this reallocation, alba, A -, female adults eclose earlier, retain more larva-derived resources in their fat bodies for somatic maintenance and for reproduction, and, in some conditions, mature their eggs faster than do aa females. Alba females are also less attractive to males than are aa females and mate less frequently. Evolutionary implications of these results are discussed.

Homothallic yeasts switch cell types (mating types a and α) at high frequency by changing the alleles of the mating type locus, MAT a and MAT α. We have proposed in the cassette model that yeast cells contain silent MAT a and MAT α blocs (“cassettes”), copies of which can be substituted at the mating type locus for the resident information. The existence of silent cassettes was originally proposed to explain efficient switching of a defective MAT α locus ( mat α) to a functional MAT α locus. We report here that this “healing” of mat mutations is a general property of the mating type interconversion system and is not specific to the class of mat α mutations studied earlier: a defective MAT a ( mat a 1 ) switches readily to MAT a and various mat α loci switch readily to MAT α. These observations satisfy the prediction of the cassette model that all mutations within MAT a and MAT α be healed. These studies also identify MAT functions that control the switching process: the same functions known to promote sporulation and prevent mating in a/α cells also inhibit the switching system in a/α cells. Finally, we present additional characterization of a natural variant of MAT α, MAT α-inc [Takano, I., Kusumi, T. & Oshima, Y. (1973) Mol. Gen. Genet . 126, 19-28] that is insensitive to switching. Our observation that MAT α-inc acts in cis suggests that it may be altered in a site concerned with excision of MAT α-inc or its replacement by another cassette.

The primary heterodiploid bacteria regenerated after Bacillus subtilis fusion, although generally noncomplementing diploids, behave in pedigree analysis as multipotential systems. Individual diploid colonies yielding complete reciprocal recombinant (RR) progeny—often accompanied by one or both parents—constitute 10-30% of the total recombinant-forming units. The RR (reciprocal for 8-11 genes) usually occur in equivalent numbers both among and within individual colonies. Novel for bacteria, they demonstrate that entire parental genomes brought together within a diploid protoplast are retained as two independent replicons able to undergo classical recombination characteristic of eukaryotic gametogenesis. Parental or recombinant genomes are also subject to multiple rounds of recombination without obligate segregation and often not reciprocal. Diploid recombinant clones, sharing streptomycin resistance but reciprocal for auxotrophic markers, have displayed a partial ability to make a facultative shift in chromosome expression. They have also produced two types of prototrophs: a stable one (presumably haploid and recombinant) and an unstable one, (diploid and temporarily complementing at low frequency). It follows that chromosome extinction may affect both parental and recombinant chromosomes and does not interfere with recombination. Analysis of the number and chromosomal distribution of crossovers in all recombinants and those from single diploid clones shows increased frequency of exchange in the regions of the replication origin and terminus, possibly a result of the association of these sites with the cell wall or membrane.

Sulfometuron methyl, a sulfonylurea herbicide, blocks growth of bacteria, yeast, and higher plants by inhibition of acetolactate synthase (EC 4.1.3.18), the first common enzyme in the biosynthesis of branched-chain amino acids. Spontaneous mutations that confer increased resistance to the herbicide were obtained in cloned genes for acetolactate synthase from Escherichia coli and Saccharomyces cerevisiae . The DNA sequence of a bacterial mutant gene and a yeast mutant gene revealed single nucleotide differences from their respective wild-type genes. The mutations result in single amino acid substitutions in the structurally homologous aminoterminal regions of the two proteins, but at different positions. The bacterial mutation results in reduced levels of acetolactate synthase activity, reduced sensitivity to sulfometuron methyl, and unaltered resistance to feedback inhibition by valine. The yeast mutation results in unaltered levels of acetolactate synthase activity, greatly reduced sensitivity to sulfometuron methyl, and slightly reduced sensitivity to valine.

By using the diffusion equation method, the level of genetic variability maintained under mutation pressure in a finite population is investigated, assuming autocorrelated random fluctuation of selection intensity. An appropriate mathematical model was formulated to treat the change of gene frequencies, incorporating mutation pressure and fluctuating selection. Extensive Monte Carlo simulation experiments were also performed to supplement the theoretical treatments. Excellent agreement between the two results suggests the validity of the present diffusion model for the autocorrelated selection. One of the most important findings from the simulation studies is that mutations and random sampling drift largely determine the level of genetic variability, and that the presence of autocorrelated selection can significantly lower genetic variability only when its strength, as measured by the cumulative variance of selection intensity, is larger than about 10 3 times the mutation rate. It is pointed out that the effects of both mutations and random sampling drift have to be incorporated in order to assess the role of various factors for the maintenance of genetic variability in natural populations.

In heterokaryons between senescent and young diploid fibroblast-like cells, dominance of the former with respect to nuclear DNA synthesis (incorporation of [ 3 H]thymidine) was demonstrated. For identification of the respective partners, double-layer autoradiography was used after the old cells were labeled with [ 3 H]methionine and the young cells were labeled with [ 14 C]thymidine. Synchrony of nuclear labeling (i.e., all nuclei in a cell labeled with [ 3 H]thymidine) was observed in the majority of di- and polykaryons during the second and third of three 24-hr periods of labeling with [ 3 H]thymidine. The results are compatible with either terminal differentiation or error theories of clonal senescence.

Mouse A9 cells, deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8), were fused with normal chick erythrocytes and selected in hypoxanthine-aminopterin-thymidine medium for cells with hypoxanthine phosphoribosyltransferase activity. Recovered hybrid cells produced the chick hypoxanthine phosphoribosyltransferase exclusively, as demonstrated by electrophoretic mobility and immunoprecipitation tests, even though no chick chromosomes or chick cell-surface antigens could be identified in the hybrids. Surprisingly, the expression of the chick hypoxanthine phosphoribosyl-transferase activity in the mouse/chick hybrids required the presence of aminopterin in the growth medium; in its absence, enzyme synthesis decreased markedly. Because of the rapid and reversible modulation of hypoxanthine phosphoribosyltransferase activity, the hybrid cells could proliferate equally well in media containing hypoxanthine-aminopterin-thymidine or 8-azaguanine. Cellular selection was definitely ruled out as a possible cause. These results confirm previous reports that specific genetic information can be selectively transferred from one cell to another of a distant species. Furthermore, they demonstrate that an avian gene, whose activity is normally expressed constitutively, can become facultative when integrated into a mammalian cell. This seems to be the first instance where heterologous gene activity has been shown to be reversibly modulated in hybrid cells.

The RAD3 gene of Saccharomyces cerevisiae is required for nicking of DNA containing pyrimidine dimers or interstrand crosslinks. We have cloned the RAD3 gene and physically mapped it to 2.6 kilobase of DNA. A DNA segment of the cloned RAD3 insert was ligated into plasmid YIp5, which transforms yeast by homologous integration, and shown to integrate at the RAD3 site in chromosome V, thus verifying the cloned DNA segment to be the RAD3 gene and not a suppressor. The RAD3 gene encodes a 2.5-kilobase mRNA, extending between the Kpn I site and the Sau 3A1/ Bam HI fusion junction in plasmid pSP10, and the direction of transcription has been determined. The 2.5-kilobase transcript could encode a protein of about 90,000 daltons. We also show the deletions of the RAD3 gene to be recessive lethals, indicating that the RAD3 gene plays an important role in other cellular processes in addition to incision of damaged DNA.

The analysis of complex genetic determinants that control the ability of a fungus to colonize its host has been impaired by the lack of sophisticated genetic tools for characterizing important pathogens. We have developed a system for the genetic transformation of Magnaporthe grisea , the causal agent of rice blast disease, to overcome this limitation. A M. grisea arginine auxotroph was shown to contain a mutation ( arg3-12 ) that abolishes ornithine carbamoyltransferase activity. M. grisea strains that contain arg3-12 were used as recipients in transformation experiments with plasmid pMA2, which carries the ArgB + gene from Aspergillus nidulans . Stable prototrophic transformants arose at a frequency of about 35 per microgram of plasmid DNA. Integration of single or multiple plasmid copies occurred at a single site in the genome of each transformant; rearrangements were often created during integration. When M. grisea genomic segments were incorporated into pMA2, the presence of any one of five different M. grisea segments did not greatly affect the efficiency of transformation. Integration via homologous recombination occurred when the donor plasmid was linearized by cleaving at a unique restriction site within the M. grisea segment.

Strategies are analyzed for maximizing the ultimate limit to truncation selection for a quantitative trait in finite populations. By using a formulation of Kimura and Crow [Kimura, M. & Crow, J. F. (1978) Proc. Natl. Acad. Sci. USA 75, 6168-6171], it is shown that the limit is maximized by truncating at the mode with the highest ordinate of the phenotypic distribution. This implies 50% selection for the normal or any unimodal symmetric distribution and, for skewed distributions, selection of more than one-half if desired phenotypes are in the long tail of the distribution, less than one-half if in the short tail. For dioecious populations, the optimal procedure requires, in addition, equal numbers recorded and selected of each sex. For monoecious populations the limit can be improved by using the best individuals mated to the next-best individuals. Limitations of the results are discussed.

The Chlamydomonas reinhardtii chloroplast mutants 18-5B and 18-7G lack both the chloroplast-encoded large subunit and nuclear-encoded small subunit of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). A chloroplast intergenic-suppression model has been postulated to account for the genetic instability of 18-5B revertants. Here, we have determined the molecular basis of the 18-5B and 18-7G mutants. They contain nonsense mutations close to the 3′ and 5′ ends of their large-subunit genes, respectively. Pulse-chase experiments revealed that the 18-5B mutant produces a truncated large subunit that is unstable. In connection with previous experiments, this work identifies nonsense suppression in the chloroplast. Small subunits are also synthesized but then degraded in the mutants. Thus, the coordinated absence of subunits is achieved through degradation of the small subunit in the specific absence of the large subunit.

A diffusion model for the joint action of genotype-dependent migration and genotype-independent population regulation is derived and investigated. The diploid, monoecious population occupies a finite chain of equally spaced colonies; generations are discrete and nonoverlapping; mating is random in each colony. A boundary-value problem is deduced for the gene frequencies at a multiallelic locus in the absence of mutation, selection, and random drift. This problem is studied for two alleles and constant and uniform population density and drift and diffusion coefficients. All equilibria are shown to be monotone, and explicit conditions for a protected polymorphism are established. Two examples of asymptotically stable clines are presented. It is demonstrated that genotype dependence of the mean displacements is necessary to produce or maintain spatial differentiation of gene frequencies.

We have previously demonstrated that translocations of V H genes from chromosome 14 to chromosome 8 and of the c- myc oncogene from chromosome 8 to chromosome 14 occur in Burkitt lymphomas with the t(8;14) chromosome translocation. An association of the c- myc gene with the C μ immunoglobulin gene has been observed in some but not all Burkitt lymphomas studied previously. In the present study, we have investigated the organization of the human heavy chain locus and of the c- myc gene in the P3HR-1 Burkitt lymphoma cell line. Becuase mouse/P3HR-1 somatic cell hybrids that retain only the 14q+ chromosome and no other human chromosome contain the human C μ and C γ genes but not V H genes, we have concluded that the breakpoint on chromosome 14 in P3HR-1 cells is distal to C μ and between C μ and V H . Thus, the breakpoint of human chromosome 14 differs in different Burkitt lymphoma cell lines. We also found that the human c- myc oncogene translocated to chromosome 14 in the P3HR-1 cell line is not recombined with the C μ gene. The breakpoint on human chromosome 8 may therefore also differ in different Burkitt lymphoma cell lines, because we have observed DNA rearrangement of the c- myc gene with the C μ gene in only some of the Burkitt lymphoma cell lines studied elsewhere. Interestingly, high levels of transcripts of the c- myc oncogene were observed in Burkitt lymphomas with translocated c- myc oncogenes both rearranged and unrearranged. Therefore, the translocation of a c- myc oncogene to the heavy chain locus on human chromosome 14 is apparently sufficient for its transcriptional activation and may be an essential step in the pathway leading to neoplasia.

If a population (species) consists of n haploid lines (subpopulations) which reproduce asexually and each of which is subject to random extinction and subsequent replacement, it is shown that, at equilibrium in which mutational production of new alleles and their random extinction balance each other, the genetic diversity (1 minus the sum of squares of allelic frequencies) is given by 2 N e v /(1 + 2 N e v ), where [Formula: see text] in which Ñ is the harmonic mean of the population size per line, n is the number of lines (assumed to be large), λ is the rate of line extinction, and v is the mutation rate (assuming the infinite neutral allele model). In a diploid population (species) consisting of n colonies, if migration takes place between colonies at the rate m (the island model) in addition to extinction and recolonization of colonies, it is shown that effective population size is [Formula: see text] If the rate of colony extinction (λ) is much larger than the migration rate of individuals, the effective population size is greatly reduced compared with the case in which no colony extinctions occur (in which case N e = nÑ ). The stepping-stone type of recolonization scheme is also considered. Bearing of these results on the interpretation of the level of genetic variability at the enzyme level observed in natural populations is discussed from the standpoint of the neutral mutation-random drift hypothesis.

Several aneuploid lines and one intervarietal substitution line of the hexaploid wheat Triticum aestivum (2 n = 6 x = 42; genomes AABBDD) cv. Chinese Spring were used to study the effects of different doses of chromosomes 1B, 1D, or 1A on the amount of the high molecular weight (“HMW”) glutenins and gliadins of endosperm. These homeologous chromosomes carry HMW glutenin and gliadin gene clusters on their long and short arms, respectively. Increasing the dosage of chromosome 1B of Chinese Spring in plants having in their 3 n endosperm zero or the normal three doses of the homeologue 1D, as well as in plants carrying in their endosperm one dose of 1B of the cultivar Timstein, had a dual effect: on one hand, a nonlinear increase in the amount of each subunit encoded by the chromosome whose dosage was elevated and, on the other hand, a compensating nonspecific decrease in the amount of other HMW glutenin and gliadin subunits encoded either by the homeoalleles on 1D or by the homoalleles on 1B of Timstein, respectively. Deletion of chromosome arm 1BL, which carries only a few HMW glutenin genes, had no significant effect on the amount of HMW glutenins encoded by 1DL and HMW gliadins encoded by 1DS and 1BS. However, deletion of 1BS or 1DS, each carrying many gliadin genes, caused a significant but nonspecific increase in the HMW glutenins and gliadins encoded by the remaining arms of 1B and 1D. The possible mechanism and evolutionary implications of gene-dosage compensation in polyploid wheat are discussed.

Increased DNA levels in centrally located endosperm nuclei are shown to be related to endosperm development in Zea mays . Mitotic activity sharply decreases in endosperm cells 10-12 days after pollination. At this time nuclear size and DNA content per nucleus (where C = haploid content) sharply increase until peak levels are reached at about 14-18 days after pollination. Mean DNA content per endosperm nucleus in strain A188 was shown by Feulgen cytophotometry to increase to about 90C by this peak stage, with the pattern being remarkably consistent over four consecutive growing seasons. Some individual nuclei achieved levels of >200C. Most other strains compared during one growing season averaged even higher peak levels of DNA per nucleus than did A188. Individual nuclei in those strains reached levels as high as 690C. A decrease in DNA level was observed in older endosperms with most strains. Endosperm mutant strains did not show a significant reduction in DNA. Opaque-2 mutants in several backgrounds achieved higher levels of DNA per nucleus. DNA levels from F 1 endosperms did not indicate heterosis. Regardless of differences in DNA content, the pattern of DNA increasing as development proceeds followed by a DNA decrease was observed for most strains. Cytological studies reveal much variation in chromatin strandedness, a maximum of three nucleoli, a maximum of three nucleolar organizer regions, and ≈30 diffuse chromatin masses in older endosperm tissue. A form of DNA amplification, perhaps polytenization, appears to be occurring during endosperm development.

The Balbiani ring (BR) 1 and BR 2 genes in Chironomus tentans are functionally related and are only expressed in the salivary gland cells. Here we reveal the principal structure of the BR 1 gene and analyze the structural and evolutionary relationship between the BR 1 and BR 2 genes. The properties of the BR 1 gene, 37 kilobases in size, are derived from the analysis of a cloned cDNA sequence, pCt 21. A considerable part of the BR 1 gene consists of one or a few blocks of a tandemly repeated 246-base-pair (bp) major repeat unit. About half of this major repeat unit is in turn built from four tandem repeats of a 33-bp sequence. This hierarchic arrangement of repetitive sequences within the BR 1 gene suggests that the gene has evolved through two major amplification steps, starting from a short primordial sequence. A similar evolutionary model has been put forward for the BR 2 gene [Sümegi, J., Wieslander, L. & Daneholt, B. (1982) Cell 30, 579-587]. The two putative primordial genes contain a similar, 102-bp-long sequence (86% nucleotide sequence homology), indicating that the BR 1 and BR 2 genes most likely arose from the same ancestor sequence. During the course of evolution the two genes diverged, mainly due to differences in the length and sequence of the gene segments involved in the two amplification steps. Moreover, at least one of the BR genes was translocated to another chromosomal locus.

In human tissues, adenosine deaminase (ADA) (adenosine aminohydrolase; EC 3.5.4.4) activity can be separated by gel electrophoresis into several isozymes. A structural gene ( ADA ) on chromosome 20 codes for the “erythrocyte” isozyme, ADA-1, which is also expressed in some nonerythroid tissues. Nonerythroid cells also differentially express five ADA “tissue isozymes” of a greater molecular weight than ADA-1. Each ADA tissue isozyme has a characteristic electrophoretic mobility and tissue distribution. It has been suggested that these ADA tissue isozymes are composed of ADA-1 and other components. We report that the expression of one of these tissue isozymes, ADA-d, is dependent upon ADA on chromosome 20 and another gene on chromosome 6 which functions in the assembly of the ADA tissue isozymes. In human-mouse hybrids segregating human chromosomes, chromosome 6 + ,20 + hybrids express both ADA-1 and ADA-d; chromosome 6 - ,20 + hybrids express only ADA-1; while 6 + ,20 - hybrids have no human ADA activity. ADA-d formation also occurs in vitro by self-assembly when an extract of human erythrocytes or chromosome 6 - ,20 + hybrids is mixed with a homogenate of chromosome 6 + ,20 - hybrids. The gene on chromosome 6, designated ADCP , codes for an adenosine d eaminase c omplexing p rotein. The product of ADCP presumably combines with ADA-1 to form the ADA tissue isozymes. The data are consistent with the hypothesis that the distribution of enzymatic activity between ADA-1 and the tissue isozymes depends on the expression of the gene for ADA complexing protein, while the differences in the electrophoretic mobilities of the ADA isozymes, except ADA-1, are generated, as suggested by others, by the degree of glycosylation of the complexing protein.

An efficient transformation system has been developed for Neurospora crassa that uses spheroplasts and pVK88 plasmid DNA. pVK88 is a recombinant Escherichia coli plasmid carrying the N. crassa qa-2 + gene which encodes catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) and is part of the qa gene cluster. The recipient strain carries a stable qa-2 - mutation and an arom-9 - mutation, thus lacking both catabolic and biosynthetic dehydroquinase activities. Transformants were selected as colonies able to grow in the absence of an aromatic amino acid supplement. These colonies were qa-2 + and had normal levels of catabolic dehydroquinase. DNA·DNA hybridization evidence with appropriate labeled probes indicates clearly that in some instances transformation involves the integration of bacterial plasmid sequences together with the qa-2 + gene into the N. crassa genome. On the basis of genetic, enzyme assay, and DNA hybridization data, at least three types of transformation events can be distinguished: ( i ) replacement of the qa-2 - gene by the qa-2 + gene without any effect on the expression of the other genes in the qa cluster, ( ii ) linked insertion of a normal qa-2 + gene accompanied by inactivation of the adjacent qa-4 + gene, and ( iii ) insertion of a normal qa-2 + gene at an unlinked site in the N. crassa genome. This newly integrated qa-2 + genetic material is inherited in a typical Mendelian fashion. A low level of transformation has also been obtained by using linear total N. crassa DNA. Two such qa-2 + transformants are unlinked to the qa-2 - gene of the recipient.

A large number of nonmotile mutants of the gliding bacterium Myxococcus xanthus have been isolated and partly characterized. About [unk] of these mutants are conditional mutants of a novel kind: mutant cells become transiently motile after contact with nonmutant cells or with cells of a different mutant type. These “stimulatable” mutants fall into five phenotypic classes (types B, C, D, E, and F). Most mutants are nonstimulatable (type A) and never become motile, but type A cells (and wild-type cells) can stimulate cells of any of the other five types. Stimulatable mutants of different types are capable of stimulating each other. For example, in a mixture of B and C cells, both become motile. Linkage analysis using a generalized transducing phage has shown that each of types B, C, D, E, and F corresponds to a single distinct genetic locus. Type A mutants, by contrast, belong to at least 17 different loci. Stimulation depends on close apposition of interacting cells, because stimulation does not occur when contact between cells is prevented. It is possible that the stimulatable mutants are defective in components of the gliding mechanism that can be exchanged between cells. Alternatively, they may be defective in a system of cell communication controlling the coordinated cell movements observed in Myxococcus .

Six inbred lines of Zea mays expressing different soluble (cytosolic) malate dehydrogenase (sMDH) zymogram phenotypes were analyzed genetically. sMDH was found to be coded for by unlinked duplicated loci in four of these inbred lines. The remaining two lines were found not to possess these duplicated loci. Furthermore, the duplicated loci, sMdh1 and sMdh2 , have been found to be located on different chromosomes: sMdh1 on chromosome 1L linked to Amp1 , and sMdh2 on chromosome 5S linked to Cat1 and Amp3 . The importance of finding sMDH encoded by duplicated loci is discussed in relation to the role of chromosomal rearrangements, the relationship between the cytoplasmic and mitochondrial enzymes, and the evolution of Z. mays .

A highly repeated DNA sequence has been isolated from the maize genome as a satellite in actinomycin D/CsCl gradients. By using maize stocks differing in their heterochromatin content we have established that the sequence is a major constituent of one class of heterochromatin, knob heterochromatin, which can occur at 23 locations in the chromosome complement. The repeating unit, of 185 base pairs, has been cloned in plasmid pBR322 and its nucleotide sequence has been determined. The presence of this DNA sequence in knob heterochromatin and its absence from centromeric, nucleolar, and B chromosome heterochromatin parallels the cytogenetic differentiation previously described for these classes of heterochromatin in maize. Because knob heterochromatin has a distinctive cytological appearance and is unique in showing neocentric activity at meiosis, its association with a particular repeated DNA sequence may reflect a functional role for the sequence in the cell cycle.

The product(s) of the Myxococcus xanthus spoC locus is required for two multicellular activities in fruiting body development, rippling and sporulation. Ripples, which are formed early in development, are spatially separated ridges of cells that move synchronously. Myxospores are heat-resistant resting cells that are formed near the end of the developmental process. To investigate the function of spoC , it was cloned in an Escherichia coli plasmid, then transferred to M. xanthus by specialized transduction with coliphage P1. The plasmid, which cannot replicate in M. xanthus , integrated into the M. xanthus chromosome, producing two copies of the spoC locus in tandem. Cells containing one copy of a mutant allele and one copy of the wild-type allele displayed the wild-type phenotype. Cells containing two different mutant alleles failed to ripple or sporulate, implying that all four independent spoC mutations are in the same gene or unit of transcription. Homozygous mutant duplications arose from constructions in which DNA from a spo + donor was transduced into a spoC recipient, or vice versa, at an average frequency of 14%, indicating that gene conversion was a frequent event.

The synthesis of carotenoids by Myxococcus xanthus requires illumination with blue light. Mutations at two loci ( carA and carR ) remove the blue-light requirement and cause constitutive production of carotenoids. Mutations at a different locus ( carB ) prevent carotenogenesis in both wild-type and constitutive mutant strains. We describe here three independent car mutations induced by insertion of Tn 5 lac , a transposon that carries a transcriptional probe for exogenous promoters. All three transposon insertions block carotenogenesis even in constitutive mutant strains. One insertion is in a previously unknown car gene and the other two are in the carB locus. One of the carB insertions expresses β-galactosidase at very low levels in the dark but is strongly activated by light. When this Tn 5 lac insertion is introduced in carA or carR mutants it expresses β-galactosidase in dark- as well as light-grown cells. We conclude that carotenogenesis in M. xanthus is activated at the level of transcription by a light-induced mechanism in which the carA and the carR loci (or their gene products) take part. The potential usefulness of M. xanthus as a simple and sensitive tool for studies in photobiology is discussed.

The yeast Saccharomyces cerevisiae changes cell types by switching the alleles of the mating type locus ( MAT ) from a to α and vice versa. In the cassette model, these switches—e.g., from a to α—occur when a replica of silent α information (an α “cassette”) replaces the resident a cassette at the mating type locus and is thereby expressed. We have identified a mutation in the locus postulated to be the silent α information ( HML α) and find that a mutation is introduced into the mating type locus as a result of interconversion: HML α - MAT α cells switch to MAT a and then to MAT α - . The MAT α - mutation leads to defective mating and behaves like some previously identified MAT α - mutations. These observations satisfy the prediction of the cassette and controlling element models that genetic information is transmitted from HML α to the mating type locus.

From a mass culture of diploid human lymphoblasts we have isolated a subline resistant to 1 μM ouabain, and from this, a subline resistant to 10 μM ouabain. These sublines occurred spontaneously, but similar mutants were induced with 10-fold increased frequency by treatment with mutagens, in which case they could be selected as clones in soft agarose. Scatchard plot analyses of ouabain binding indicated that the subline resistant to 1 μM ouabain retained an average of 39% of the high-affinity ouabain receptors of the parental lymphoblast line, and the subline resistant to 10 μM ouabain retained an average of 8.4%. The ouabain binding site is known to be located in the Na + ,K + -ATPase (ATP phosphohydrolase, EC 3.6.1.3) molecule, an essential cell membrane enzyme that mediates ion transport. Studies on Na + ,K + -ATPase activity, using 86 Rb in the absence and presence of ouabain, indicated that our parental lymphoblasts contained one population of Na + ,K + -ATPase molecules highly sensitive to ouabain inhibition, the subline resistant to 10 μM ouabain contained one population relatively insensitive to ouabain, and the subline resistant to 1 μM ouabain contained both populations. Thus, the moderately resistant subline appears to be heterozygous for ouabain resistance, probably containing a structural mutation in the ouabain receptor region of the Na + ,K + -ATPase molecule in one of the two homologous loci for this enzyme, whereas the highly resistant subline derived from it appears to be homozygous, containing an additional mutation in the other Na + ,K + -ATPase locus.

Transfer RNA 5 Lys was isolated and purified from Drosophila melanogaster . It was iodinated in vitro with sodium [ 125 I]iodide and hybridized in situ to the polytene salivary gland chromosomes from Drosophila. Autoradiographs exposed three to four weeks allowed the localization of this tRNA to the right arm of chromosome 2 between bands 48F and 49A.

The transposon Tn 5 , which carries a gene for kanamycin resistance, can be introduced into Myxococcus xanthus , an organism that undergoes a primitive cycle of development, from Escherichia coli by the specialized transducing phage P1::Tn 5 . Tn 5 DNA sequences, but no P1 sequences, are found in the stable kanamycin-resistant transductants. Tn 5 transposes from P1 to many different chromosomal sites in Myxococcus . In each independent transductant of Myxococcus examined, the Tn 5 element is found in a different DNA fragment produced by cleaving cell DNA with a restriction endonuclease. Moreover, different Tn 5 insertions have been found linked to the first 20 different genetic sites tested. Once inserted into the Myxococcus chromosome, Tn 5 remains fixed in position during growth and when transferred to another strain by generalized transduction. To analyze developmental or other mutants that have no selectable phenotype themselves, a general method has been devised, and tested, for the systematic isolation of a Tn 5 insertion near any arbitrary locus.

On purification of human adenovirus type 12 (Ad12) by equilibrium sedimentation in CsCl density gradients, two bands of particles, Ad12-3 and Ad12-3a, are observed. The particles from band Ad12-3a contain a recombinant of human host cell DNA and of Ad12 DNA. The human cell DNA sequences contain repetitive DNA recurring 200 to 500 times in cellular DNA. Ad12 DNA and the recombinant genomes exhibit the same or similar lengths. This finding suggests that a constant amount of DNA is packaged into complete Ad12 particles. On cleavage of KB cellular DNA with Eco RI, Bam HI, Hin fI, Msp I, Mbo I Pst I, or Bgl II, the 32 P-labeled cellular DNA from Ad12-3a particles hybridizes on Southern blots to distinct bands of KB DNA. There is also less-specific background hybridization that is not observed in the control. The cellular DNA from Ad12-3a particles is not methylated, whereas the same cellular sequences in KB cell DNA appear to be extensively methylated. On denaturation and renaturation, the recombinant DNA molecules are converted to molecules half as long as Ad12 DNA, as determined by gel electrophoresis and electron microscopy. The recombinant DNA molecules were terminally labeled by exonuclease III treatment and subsequent refilling of the depleted segments with [ 32 P]dNTPs by using DNA polymerase I (Klenow fragment). When these molecules were cleaved with Eco RI, Bam HI, Msp I, or Pst I, only one terminal DNA fragment was found to be labeled. The results of partial digestion experiments using Msp I, Hin fI, or Mbo I are consistent with a model in which 700-1150 base pairs from the left terminus of Ad12 DNA are linked to host cell DNA containing repetitious sequences, and this structure is symmetrically duplicated as a large inverted repeat of the type ABCDD′C′B′A′. The Ad12 DNA sequences are flanking the entire molecule, which consists mainly of human KB cell DNA. The recombinants appear to be stable on serial passage of the virus preparation for many years, although variations in the sequence of the recombinants occur. These symmetric recombinant (SYREC) molecules suggest a way to use adenovirus DNA as a eukaryotic vector. Their occurrence provides further evidence for the generation of virus-host DNA recombinants and may help elucidate the role this interaction may have in adenovirus replication and oncogenesis.