Uncontrolled growth associated with novel somatic recombination in the fungus Schizophyllum

In the bracket mushroom, Schizophyllum commune, a recessive genetic alteration, mnd, causes abnormally hyperplastic three-dimensional mounds of hyphae to rise from the surface of both haploid and dikaryotic mycelia. mnd, although not a genetic block in the fruiting body developmental pathway, is at least partially epistatic to fruiting. Within dikaryons containing both mutant and wild-type nuclei, [mnd + mnd⁺], a nonreciprocal somatic recombination event can lead to stable conversion of the mnd⁺ region of the wild-type nucleus to mnd. This transformation to the homoallelic [mnd + mnd] condition involves no genomic areas other than the mnd region and permanently prevents any further fruiting. Studies relating to the recombination mechanism have ruled out a diploid intermediate state and other concomitants of orthodox somatic recombination, as well as whole chromosome transfer. Instead, a novel form of internuclear genetic transfer is postulated whereby a nearby locus, mob⁺, controls the mobilization of the mnd chromosomal region alone from one nucleus to the other within the binucleate cells of dikaryotic mycelia.     

Internuclear Genetic Transfer in Vegetative Dikaryons of SCHIZOPHYLLUM COMMUNE: I. Di-Mon Mating Analysis

A series of hemi-compatible dikaryon x monokaryon (di-mon) matings was designed to determine whether there was any genetic interaction between the dikaryotic nuclei. One of the nuclei in each dikaryon was known to carry a recessive gene (mnd) that promoted the development of an abnormal growth form, mound. Dikaryons containing both mnd ⁺ and mnd nuclei produced mosaic colonies that consisted of three distinct kinds of hyphae: mound, fruiting body, and vegetative (devoid of any multihyphal structure). When dikaryotic hyphae from each of these morphological regions were used in di-mon matings, the genetic and developmental characteristics of the selected nuclear types were examined in the resulting derived dikaryons. The results showed that fruiting-body and vegetative cells contained the expected mnd and mnd⁺ nuclei. Dikaryotic mound hyphae, however, contained only mnd nuclei. In a manner as yet unresolved, but clearly dependent on the presence of the mnd allele, the mnd ⁺ allele of a wild nucleus was altered to, or acquired, the mnd allele. A number of hypotheses were considered to explain the genetic event(s) that generates [mnd + mnd*] dikaryotic cells from [mnd⁺ + mnd] cells, but none was found to be entirely satisfactory.     

Internuclear Genetic Transfer in Dikaryons of SCHIZOPHYLLUM COMMUNE. II. Direct Recovery and Analysis of Recombinant Nuclei

The recessive gene, mound (mnd), allows the appearance of globose masses of compacted hyphae. Dikaryons of Schizophyllum commune that are heteroallelic for mnd [(mosaic dikaryons: (mnd + mnd(+))] have been successfully dedikaryotized in cholate-containing medium in order to recover the component nuclear types directly. The relative proportion of the two recovered monokaryotic types shows in all cases a marked deviation from 1:1. Hyphae from nonmound mycelial regions yield monokaryotic types identical to those originally used to form the dikaryons. In hyphae from mound-forming regions, however, homoallelism of the mnd allele has been demonstrated; the nuclear type that formerly contained the mnd(+) allele acquired a mnd allele.-The process of internuclear transfer or recombination is unaccompanied by the simultaneous alteration of any additional genetic markers carried by the recipient nucleus. The newly acquired mnd allele segregates in Mendelian fashion in subsequent outcrosses and appears to be chromosomally located. A novel process of somatic recombination, with several features distinct from classical parasexual mitotic recombination, appears to be in operation.     

Probable exclusion of the cortexin-encoding gene as a candidate for mouse neurological mutants: nervous,tottering and motor neuron degeneration

We have tested the gene encoding cortexin, Ctxn, which maps to chromosome 8, as a candidate for the mouse neurological mutants: nervous (nr), tottering (tg) plus tottering-leaner (tg^la), and motor neuron degeneration (mnd) by Northern blot analysis of brain poly(A)⁺ RNA and direct polymerase chain reaction (PCR) sequencing. No difference from wild-type was seen in any of these mutants. Based upon these observations, we conclude that Ctxn is not involved in the genetic defects found in nr, tg or mnd mice.     

Regulation of the plastochron by three many-noded dwarf genes in barley

The plastochron, the time interval between the formation of two successive leaves, is an important determinant of plant architecture. We genetically and phenotypically investigated many-noded dwarf (mnd) mutants in barley. The mnd mutants exhibited a shortened plastochron and a decreased leaf blade length, and resembled previously reported plastochron1 (pla1), pla2, and pla3 mutants in rice. In addition, the maturation of mnd leaves was accelerated, similar to pla mutants in rice. Several barley mnd alleles were derived from three genes—MND1, MND4, and MND8. Although MND4 coincided with a cytochrome P450 family gene that is a homolog of rice PLA1, we clarified that MND1 and MND8 encode an N-acetyltransferase-like protein and a MATE transporter-family protein, which are respectively orthologs of rice GW6a and maize BIGE1 and unrelated to PLA2 or PLA3. Expression analyses of the three MND genes revealed that MND1 and MND4 were expressed in limited regions of the shoot apical meristem and leaf primordia, but MND8 did not exhibit a specific expression pattern around the shoot apex. In addition, the expression levels of the three genes were interdependent among the various mutant backgrounds. Genetic analyses using the double mutants mnd4mnd8 and mnd1mnd8 indicated that MND1 and MND4 regulate the plastochron independently of MND8, suggesting that the plastochron in barley is controlled by multiple genetic pathways involving MND1, MND4, and MND8. Correlation analysis between leaf number and leaf blade length indicated that both traits exhibited a strong negative association among different genetic backgrounds but not in the same genetic background. We propose that MND genes function in the regulation of the plastochron and leaf growth and revealed conserved and diverse aspects of plastochron regulation via comparative analysis of barley and rice.     

Rate of Accumulation of Luxol Fast Blue Staining Material and Mitochondrial ATP Synthase Subunit 9 in Motor Neuron Degeneration Mice

The rate of accumulation of Luxol Fast Blue staining material in the hippocampus of motor neuron degeneration (mnd/mnd) mice, a model of Batten Disease, was quantitated. Stained material increased linearly up to 8 months of age. A quantitative immunoassay was used to measure levels of mitochondrial ATP synthase subunit 9 in brain and liver of mnd/mnd mice. Levels of subunit 9 increased progressively throughout the lifespan of mnd/mnd mice reaching levels approximately 5-fold higher than in control animals. The rate of accumulation of subunit 9 is not consistent with any simple complete or partial degradation defect that is constant throughout the animal's life. Two more complicated models are discussed which are consistent with the observed accumulation rate of subunit 9.     

Abnormalities in mitochondria-associated membranes and phospholipid biosynthetic enzymes in the mnd/mnd mouse model of neuronal ceroid lipofuscinosis

Endoplasmic reticulum-like membranes (MAM) that are associated with mitochondria have been implicated as intermediates in the import of lipids, particularly phosphatidylserine, from the endoplasmic reticulum to mitochondria (Vance, J.E. (1990) J. Biol. Chem. 265, 7248–7256; Shiao, Y.-J. et al. (1995) J. Biol. Chem. 270, 11190–11198). We have now examined further the role of MAM in lipid metabolism using the mnd/mnd mouse, a model for the human degenerative disease neuronal ceroid lipofuscinosis. The biochemical phenotype of the mnd/mnd mutant mouse (in which lipids and proteins accumulate abnormally in storage bodies in cells of affected tissues) suggested that the mutation might lead to impaired mitochondrial import of lipids and proteins as a result of a defective linkage between MAM and mitochondria. We, therefore, investigated the status of MAM and phospholipid metabolism in mnd/mnd mice livers. Separation of MAM from livers of older, but not younger, mnd/mnd mice was aberrant. In addition, the amount of the MAM-specific protein, phosphatidylethanolamine N-methyltransferase-2 (PEMT2), was greatly reduced in homogenates and MAM from livers of mnd/mnd mice of all ages, although PEMT2 mRNA abundance was normal. Moreover, PEMT activity in MAM from mnd/mnd mice was 60% less than in control mice. Activities of two additional phospholipid biosynthetic enzymes — CTP:phosphocholine cytidylyltransferase and phosphatidylserine synthase — were also reduced by >50% in mnd/mnd microsomes. Radiolabeling experiments in hepatocytes indicated that neither the mitochondrial import nor the subsequent metabolism of phosphatidylserine was grossly affected in mnd/mnd mice. However, 3 proteins (cytochrome b₅, NADH:cytochrome b₅ reductase and mitochondrial F₁F₀-ATP synthase c subunit) which are normally present in mitochondria were partially redistributed to microsomes in mnd/mnd mouse liver. These studies indicate that MAM are defective in the mnd/mnd mutant mouse in which the biochemical phenotype includes an abnormal accumulation of lipids and proteins in storage bodies.     

Cloning and Identification of Conjugative Transfer Origins in the Rhizobium meliloti Genome

A simple approach was used to identify Rhizobium meliloti DNA regions with the ability to convert a nontransmissible vector into a mobilizable plasmid, i.e., to contain origins of conjugative transfer (oriT, mob). RecA-defective R. meliloti merodiploid populations, where each individual contained a hybrid cosmid from an R. meliloti GR4 gene library, were used as donors en masse in conjugation with another R. meliloti recipient strain, selecting transconjugants for vector-encoded antibiotic resistance. Restriction analysis of cosmids isolated from individual transconjugants resulted in the identification of 11 nonoverlapping DNA regions containing potential oriTs. Individual hybrid cosmids were confirmed to be mobilized from the original recA donors at frequencies ranging from 10⁻² to 10⁻⁵ per recipient cell. DNA hybridization experiments showed that seven mob DNA regions correspond to plasmid replicons: four on symbiotic megaplasmid 1 (pSym1), one on pSym2, and another two on each of the two cryptic plasmids harbored by R. meliloti GR4. Another three mob clones could not be located to any plasmid and were therefore preliminarily assigned to the chromosome. With this strategy, we were able to characterize the oriT of the conjugative plasmid pRmeGR4a, which confirmed the reliability of the approach to select for oriTs. Moreover, transfer of the 11 mob cosmids from R. meliloti into Escherichia coli occurred at frequencies as high as 10⁻¹, demonstrating the R. meliloti gene transfer capacity is not limited to the family Rhizobiaceae. Our results show that the R. meliloti genome contains multiple oriTs that allow efficient DNA mobilization to rhizobia as well as to phylogenetically distant gram-negative bacteria.     

Compétitivité pour l'infection entre souches deRhizobium meliloti: Role de la mobilité

Resumé A partir de la souche deR. meliloti Ve 26 mobile et chimiotactique, un mutant non mobile (mob⁻) et un mutant hyper-mobile (mob⁺) ont été isolés. L'importance du r?le de la mobilité dans les phénomènes d'infection et de nodulation lors d'inoculations mixtes réalisées en miniserre expérimentale, est montrée: le mutant Ve 26 mob⁺ forme 86% et 48% de nodules respectivement quand il est inoculé en mélange avec le mutant Ve 26 mob⁻ ou avec la souche 2011 (non mobile par rapport au mutant Ve 26 mob⁺). L'inoculation mixte avec le mutant Ve 26 mob⁻ et la souche 2011 deR. meliloti, en utilisant différents rapports de concentrations, a permis de montrer qu'il existe entre ces souches un effet antagoniste pour l'infection.      

A novel fission yeast gene, kms1 +, is required for the formation of meiotic prophase-specific nuclear architecture

In the meiotic prophase nucleus of the fission yeast Schizosaccharomyces pombe, chromosomes are arranged in an oriented manner: telomeres cluster in close proximity to the spindle pole body (SPB), while centromeres form another cluster at some distance from the SPB. We have isolated a mutant, kms1, in which the structure of the meiotic prophase nucleus appears to be distorted. Using specific probes to localize the SPB and telomeres, multiple signals were observed in the mutant nuclei, in contrast to the case in wild-type. Genetic analysis showed that in the mutant, meiotic recombination frequency was reduced to about one-quarter of the wild-type level and meiotic segregation was impaired. This phenotype strongly suggests that the telomere-led rearrangement of chromosomal distribution that normally occurs in the fission yeast meiotic nucleus is an important prerequisite for the efficient pairing of homologous chromosomes. The kms1 mutant was also impaired in karyogamy, suggesting that the kms1 ⁺ gene is involved in SPB function. However, the kms1 ⁺ gene is dispensable for mitotic growth. The predicted amino acid sequence of the gene product shows no significant similarity to known proteins.     

Possibilities of the conjugation process in mycobacteria

Results obtained when studying conjugation in mycobacteria by means of different methods are summarized. The method of conjugation on surface of a solid complete medium was tested with different auxotrophic mutants of different strains ofMycobacterium smegmatis. It was not possible to obtain positive results even by means of the above method. This was probably due to unsuitability of the chosen strains ofMycobacterium smegmatis. Preparation of the donor strain by transfer of the F factor fromEscherichia coli F’ORF 1ade ⁺ lac⁺ pro⁺ toMycobacterium phlei PA adeStm ^r by means of sexduction is described. Frequency of the phenotype PAade ⁺ Stm^r increased in the average by two and a half orders of magnitude with respect to the control, however, a further transfer from cultures of the cellsade ⁺ Stm^r to cells ade could not be demonstrated. Experiments aimed at transferring the R factor from strainsEscherichia coli K-12 toMycobacterium phlei were unsuccessful.     

A Study of Rdna Magnification Phenomenon in a Repair-Recombination Deficient Mutant of DROSOPHILA MELANOGASTER

The rDNA magnification process consists of a rapid and inheritable rDNA increase occurring in bobbed males: in a few generations the bb loci acquire the wild-type rDNA value and reach a bb⁺ phenotype.—We have analyzed the rDNA magnification process in the repair-recombination-deficient mutant mei9^a, both at the phenotypical and rDNA content levels. In mei9^a bb double mutants the recovery of bb⁺ phenotype is strongly disturbed and the rDNA redundancy value fails to reach the wild-type level. The strong effect of this meiotic mutation on rDNA magnification suggests a close relationship between this phenomenon and the repair-recombination processes.     

Characterization of the ColE1 mobilization region and its protein products

Summary A third of the 6.6 kb genome of ColE1 is devoted to mobilization (mob) genes necessary to promote its specific transfer in the presence of conjugative plasmids. Themob region is genetically complex: twomob genes are entirely overlapped by a third. Oligonucleotide-directed mutagenesis was used to insert an amber codon into one of the overlapped genes and make possible a full complementation analysis ofmob. Fourmob genes essential for mobilization by R64drd11 were thus identified. Fragments ofmob were subcloned under control of the P_tac promoter in a suitable vector, overexpressed in minicells and the mobilization proteins visualized. A comprehensive alignment of themob region of ColE1 with those of its close relatives ColK and ColA demonstrating that the four essentialmob genes are conserved is also presented.     

Construction of Streptococcus lactis subsp. lactis Strains with a Single Plasmid Associated with Mucoid Phenotype

Lactose-fermenting mucoid (Lac⁺ Muc⁺) variants of plasmid-free Streptococcus lactis subsp. lactis MG1614 were obtained by protoplast transformation with total plasmid DNA from Muc⁺S. lactis subsp. cremoris ARH87. By using plasmid DNA from these variants for further transformations followed by novobiocininduced plasmid curing, Lac⁻ Muc⁺ MG1614 strains containing only a single 30-megadalton plasmid could be constructed. This plasmid, designated pVS5, appeared to be associated with the Muc⁺ phenotype.     

Members of the pogo superfamily of DNA-mediated transposons in the human genome

Bacillus subtilis, likeEscherichia coli, possesses several sets of genes involved in the utilization ofβ-glucosides. InE. coli, all these genes are cryptic, including the genes forming thebgl operon, thus leading to a Bgl^? phenotype. We screened forB. subtilis chromosomal DNA fragments capable of reverting the Bgl⁺ phenotype associated with anE. coli hns mutant to the Bgl^? wild-type phenotype. OneB. subtilis chromosomal fragment having this property was selected. It contained a putative Ribonucleic AntiTerminator binding site (RAT sequence) upstream from thebglP gene. Deletion studies as well as subcloning experiments allowed us to prove that the putativeB. subtilis bglP RAT sequence was responsible for the repression of theE. coli bgl operon. We propose that this repression results from the titration of the BglG antiterminator protein ofE. coli bgl operon by our putativeB. subtilis bglP RAT sequence. Thus, we report evidence for a new cross interaction between heterologous RAT-antiterminator protein pairs.     

The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5′ and 3′ untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

Germination and growth ofErythrorchis ochobinesis (Orchidaceae) accelerated by monokaryons and dikaryons ofLenzites betulinus andTrametes hirsuta

Four sib-monokaryons and two reconstituted dikaryons of two basidiomycetes,Lenzites betulinus andTrametes hiruta, accelerated the seed germination ofErythrorchis ochobiensis, an achlorophyllous orchid. All isolates ofL. betulinus and three isolates ofT. hirsuta induced the development of plants from germinated seeds. Although three monokaryotic isolates ofT. hirsuta failed to induce the development of plants, the reconstituted dikaryons induced the development.     

Involvement of a Plasmid in Production of Ropiness (Mucoidness) in Milk Cultures by Streptococcus cremoris MS

Curing and genetic transfer experiments showed that lactose-fermenting ability (Lac⁺) and the ability to produce mucoidness in milk cultures (Muc⁺) in Streptococcus cremoris MS were coded on plasmids. The Lac⁺ phenotype was associated with a 75.8-megadalton plasmid, pSRQ2201. The Muc⁺ phenotype was associated with a 18.5-megadalton plasmid, pSRQ2202. The Lac plasmid, pSRQ2201, was first conjugatively transferred from S. cremoris MS to Lac⁻S. lactis ML-3/2.2. Later, the Muc plasmid, pSRQ2202, was conjugatively transferred from Lac⁻ Muc⁺S. cremoris MS04 to Lac⁺ nonmucoid S. lactis transconjugant ML-3/2.201. Subsequently, pSRQ2201 and pSRQ2202 were cotransferred from Lac⁺ Muc⁺S. lactis transconjugant ML-3/2.202 to Lac⁻, nonmucoid, malty S. lactis 4/4.2 and S. lactis subsp. diacetylactis SLA3.25. Transconjugants showing pSRQ2201 were Lac⁺; those containing pSRQ2202 were Muc⁺. With the transfer of pSRQ2202, the transconjugants S. lactis ML-3/2.202 and S. lactis subsp. diacetylactis SLA3.2501 not only acquired the Muc⁺ phenotype but also resistance to bacteriophages, which were lytic to the respective parent strains S. lactis ML-3/2.201 and S. lactis subsp. diacetylactis SLA3.25.