The maize viviparous15 locus encodes the molybdopterin synthase small subunit

A new Zea mays viviparous seed mutant, viviparous15 (vp15), was isolated from the UniformMu transposon-tagging population. In addition to precocious germination, vp15 has an early seedling lethal phenotype. Biochemical analysis showed reduced activities of several enzymes that require molybdenum cofactor (MoCo) in vp15 mutant seedlings. Because MoCo is required for abscisic acid (ABA) biosynthesis, the viviparous phenotype is probably caused by ABA deficiency. We cloned the vp15 mutant using a novel high-throughput strategy for analysis of high-copy Mu lines: We used MuTAIL PCR to extract genomic sequences flanking the Mu transposons in the vp15 line. The Mu insertions specific to the vp15 line were identified by in silico subtraction using a database of MuTAIL sequences from 90 UniformMu lines. Annotation of the vp15-specific sequences revealed a Mu insertion in a gene homologous to human MOCS2A, the small subunit of molybdopterin (MPT) synthase. Molecular analysis of two allelic mutations confirmed that Vp15 encodes a plant MPT synthase small subunit (ZmCNX7). Our results, and a related paper reporting the cloning of maize viviparous10, demonstrate robust cloning strategies based on MuTAIL-PCR. The Vp15/CNX7, together with other CNX genes, is expressed in both embryo and endosperm during seed maturation. Expression of Vp15 appears to be regulated independently of MoCo biosynthesis. Comparisons of Vp15 loci in genomes of three cereals and Arabidopsis thaliana identified a conserved sequence element in the 5' untranslated region as well as a micro-synteny among the cereals.     

The use of compound heterozygotes and Hprt selection to analyze X-linked mottled alleles associated with prenatal lethality

X-linked mutant alleles associated with prenatal male lethality are difficult to analyze because only heterozygous females are readily available for study. Genomic analysis of the mutant allele is facilitated by the construction of somatic cell hybrids because this enables the segregation of the X Chromosomes (Chrs) that carry the mutant and wild-type alleles. We describe here a method that ensures that the X Chr carrying the mutant allele is retained in somatic cell hybrids in an active selectable state. This is achieved by mating heterozygous females to males that carry a mutation at the hypoxanthine phosphoribosyl transferase (Hprt) locus. The resultant F₁ females are compound heterozygotes, and when cells from these females are fused to HPRT− Chinese hamster cells and subjected to selection in HAT medium, the only survivors are those hybrid cells that retain an active X Chr carrying the mutant allele together with the wild-type Hprt allele. We use hybrids constructed by this method to demonstrate that there are no gross deletions or genomic rearrangements present in three mottled alleles associated with prenatal male lethality. Received: 8 January 1996 / Accepted: 29 February 1996     

Regulation of programmed cell death in maize endosperm by abscisic acid

Cereal endosperm undergoes programmed cell death (PCD) during its development, a process that is controlled, in part, by ethylene. Whether other hormones influence endosperm PCD has not been investigated. Abscisic acid (ABA) plays an essential role during late seed development that enables an embryo to survive desiccation. To examine whether ABA is also involved in regulating the onset of PCD during endosperm development, we have used genetic and biochemical means to disrupt ABA biosynthesis or perception during maize kernel development. The onset and progression of cell death, as determined by viability staining and the appearance of internucleosomal DNA fragmentation, was accelerated in developing endosperm of ABA-insensitive vp1 and ABA-deficient vp9 mutants. Ethylene was synthesized in vp1 and vp9 mutant kernels at levels that were 2–4-fold higher than in wild-type kernels. Moreover, the increase and timing of ethylene production correlated with the premature onset and accelerated progression of internucleosomal fragmentation in these mutants. Treatment of developing wild-type endosperm with fluridone, an inhibitor of ABA biosynthesis, recapitulated the increase in ethylene production and accelerated execution of the PCD program that was observed in the ABA mutant kernels. These data suggest that a balance between ABA and ethylene establishes the appropriate onset and progression of programmed cell death during maize endosperm development.     

A homozygous nonsense mutation in SOX9 in the dominant disorder campomelic dysplasia: a case of mitotic gene conversion

Campomelic dysplasia (CD; MIM 114290), an autosomal dominant skeletal malformation syndrome with XY sex reversal, is caused by heterozygous de novo mutations in and around the SOX9 gene on 17q. We report a patient with typical signs of CD, including sex reversal, who was, surprisingly, homozygous for the nonsense mutation Y440X. Since neither parent carried the Y440X mutation, possible mechanisms explaining the homozygous situation were a de novo mutation followed by uniparental isodisomy, somatic crossing over, or gene conversion. As the patient was heterozygous for six microsatellite markers flanking SOX9, uniparental isodisomy and somatic crossing over were excluded. Analysis of intragenic single-nucleotide polymorphisms suggested that the homozygous mutation arose by a mitotic gene conversion event involving exchange of at least 440 nucleotides and at most 2,208 nucleotides between a de novo mutant maternal allele and a wild-type paternal allele. Analysis of cloned alleles showed that homozygous mutant cells constituted about 80% of the leukocyte cell population of the patient, whereas about 20% were heterozygous mutant cells. Heterozygous Y440X mutations, previously described in three CD cases, have been identified in seven additional cases, thus constituting the most frequent recurrent mutations in SOX9. These patients frequently have a milder phenotype with longer survival, possibly because of the retention of some transactivation activity of the mutant protein on SOX9 target genes, as shown by cell transfection experiments. The fact that the patient survived for 3 months may thus be explained by homozygosity for a hypomorphic rather than a complete loss-of-function allele, in combination with somatic mosaicism. This is, to our knowledge, the first report of mitotic gene conversion of a wild-type allele by a de novo mutant allele in humans.     

The viviparous8 mutation delays vegetative phase change and accelerates the rate of seedling growth in maize

Post-embryonic shoot development in plants can be divided into a juvenile vegetative, an adult vegetative, and a reproductive phase, which are expressed in different domains on the shoot axis. The number and position of the phytomers in each phase are determined by the time at which a plant begins and ceases making phytomers of a particular phase and the rate at which phytomers are made during that phase. The viviparous8 (vp8) mutation of maize increases the number of juvenile vegetative phytomers and decreases the number of adult vegetative phytomers by affecting both of these processes. vp8 increases the number of juvenile vegetative phytomers by increasing the rate of leaf initiation early in shoot development and delaying the juvenile-to-adult transition (vegetative maturation). It reduces the number of adult phytomers because the delay in vegetative maturation is not matched by a corresponding delay in flowering time; vp8 plants produce a tassel at the same time as wild-type plants. Thus, Vp8 normally controls the production of a factor that functions both to repress the rate of growth early in shoot development and to promote vegetative maturation, but which has no major role in floral induction. vp8 dramatically enhances the phenotypes of the dwarf and Teopod mutants and requires a functional Glossy15 gene to prolong the expression of juvenile epidermal traits. Evidence suggesting that vp8 does not affect phase change by reducing the level of abscisic acid is discussed.     

Introduction of <Emphasis Type="Italic">tau</Emphasis> Mutation into Cultured Rat1-R12 Cells by Gene Targeting,Using Recombinant Adeno-Associated Virus Vector

We aim to develop a cultured cell model, to serve as a system with which the altered circadian phenotypes produced by the clock gene variations could be studied in vitro. Tau mutation, which shortens the circadian period of hamsters and mice, was introduced into the CK1ε locus of cultured Rat1-R12 cells by gene targeting mediated by a recombinant adeno-associated virus (rAAV) vector. After transduction of Rat1-R12 cells with rAAV, about 0.14% of the drug-resistant cells underwent gene targeting at CK1ε locus. Of the three clones isolated, only one carried the targeted allele of tau mutation and two carried the targeted wild-type allele. The clone with the targeted tau mutant allele exhibited a significantly shorter circadian period compared to the clone with targeted wild-type allele. rAAV-mediated gene targeting in cultured somatic cells is a convenient and powerful tool for analyzing the phenotypic outcome of clock gene variations, and for elucidating the pathogenesis of the disorders associated with abnormal circadian rhythmicity.     

Absence of somatic mosaicism in 17 families with hemophilia B: an analysis with a sensitivity 10- to 1000-fold greater than that of sequencing gels

Most estimates of germ-line mosaicism have been derived from families in which there has been transmission of a mutated allele to two or more children by an unaffected individual. Previously, analyses for somatic mosaicism detected five such individuals by PCR-based sequencing and haplotype analysis at a sensitivity of approximately 1 mutant per 10 wild-type alleles. To determine whether mutations that occur later in embryogenesis also give rise to somatic mosaicism, we analyzed leukocyte DNA from 17 individuals in whom a mutation in the factor IX gene was known to have originated. Methods capable of detecting 1 mutant allele in 100–10 000 were utilized, and no further examples of somatic mosaicism were detected. If confirmed by future studies, the paucity of somatic mosaicism with mutant:wild-type allele frequencies ranging from 1:10 to 1:1000 (relative to the 11% of somatic mosaicism detected with mutant:wild-type allele frequencies of 1:1 to 1:10) may reflect a higher mutation rate and/or germ-line lineage allocation very early in embryogenesis. Received: 14 July 1995 / Revised: 1 April 1996     

SPA1 and DET1 act together to control photomorphogenesis throughout plant development

The COP1/SPA complex and DET1 function to suppress photomorphogenesis in dark-grown Arabidopsis seedlings. Additionally, they inhibit flowering under non-inductive short-day conditions. The COP1/SPA complex and DET1, as part of the CDD complex, represent distinct high-molecular-weight complexes in Arabidopsis. Here, we provide genetic evidence that these complexes co-act in regulating plant development. We report the isolation of a spa1 enhancer mutation that represents a novel, very weak allele of det1. This det1 ^esp1 mutation caused no detectable mutant phenotype in the presence of wild-type SPA1, but showed strongly synergistic genetic interaction with the spa1 mutation in the control of seedling photomorphogenesis, anthocyanin accumulation, plant size as well as flowering time. On the biochemical level, the det1 ^esp1 spa1 double mutant showed higher HY5 protein levels than either single mutant or the wild type. The genetic interaction of spa1 and det1 mutations was further confirmed in the spa1 det1-1 double mutant which carries a strong allele of det1. Taken together, these results show that SPA1 and DET1 act together to control photomorphogenesis throughout plant development. Hence, this suggests that COP1/SPA complexes and the CDD complex co-act in controlling the protein stability of COP1/SPA target proteins.     

Viviparous-1 mutation in maize conditions pleiotropic enzyme deficiencies in the aleurone

The viviparous-1 (vp1) mutation in maize (Zea mays L.) conditions a unique pleiotropic phenotype: premature germination of the embryo and failure to synthesize anthocyanin (flavonoid) pigments in the aleurone. By using a B-A translocation, it is possible to analyze the basis for the anthocyaninless phenotype of vp1 in the absence of vivipary. Anthocyaninless vp1 aleurones were found to be deficient in at least three enzymes of flavonoid biosynthesis (phenylalanine ammonia lyase, chalcone synthase, and UDPG-flavonoid glucosyltransferase) as well as in several other metabolically unrelated enzymes that show pronounced increases in late stages of aleurone development. The set of structural genes encoding such enzymes is postulated to be under the regulation of the vpl gene.     

Genetic and phenotypic characterization of cop1 mutants of Arabidopsis thaliana

Ten Arabidopsis lines that carry recessive mutations in the cop1 (constitutively photomorphogenic) locus have been isolated. These lines define at least four different alleles. All of the mutant lines produce dark-grown seedlings that mimic wild-type seedlings grown in the light. The phenotype of the dark-grown mutant seedlings includes: short hypocotyls, open and enlarged cotyledons, accumulation of anthocyanin, cell-type differentiation and chloroplast-like plastid differentiation in cotyledons. Moreover, in more prolonged dark-growth periods the mutants exhibit true leaf development that parallels that in light-grown siblings. The four mutant alleles represent two types of mutations: three alleles (cop 1-1, cop 1-2, and cop 1-3) have severely affected phenotypes whereas one allele (cop 1-4) has a less severe phenotype. Compared to the severe alleles, the cop 1-4 mutant has slightly longer hypocotyls in dark-grown seedlings and does not accumulate abnormal levels of anthocyanin. The cop1–1/cop1-4 hybrid seedlings are intermediate in many physiological properties under both dark- and light-growth conditions, relative to the two parents. These results may suggest that the extent of residual cop1 gene activity in the mutants dictates the degree to which the aberrant plant phenotype is expressed. Analysis of plants carrying both cop1 and hy, a mutation that results in a deficiency of active phyto-chrome, suggests that the cop1 gene product acts downstream of phytochrome. The differentiation of chloroplasts in the roots of light-grown cop1 plants but not in wild-type plants suggests that the wild-type cop1 gene product also normally plays a role in suppressing chloroplast development in the roots of light-grown plants. To aid the eventual molecular cloning of the cop1 locus, its chromosomal location has been mapped and a molecular marker that is located about 1 centimorgan away from the cop1 locus obtained.     

Isolation and characterization of herC, a mutation of Escherichia coli affecting maintenance of ColE1

Summary Two modes of ColE1 DNA replication are known, one dependent on RNase H, and the other RNase H independent. The cer114 mutant of the ColE1 replicon is defective in both modes and carries a single base pair alteration 95 by upstream of the replication origin. An Escherichia coli mutant which restored maintenance of the cer114 replicon was isolated. This host suppressor mutant is defective in RNase H and carries a herC, mutation located at 62 min of the E. coli chromosome. The herC, mutation is recessive to its wild-type allele and supports maintenance of the mutant replicon in the absence of RNase H. The herC, mutation alone conferred cold-sensitive growth, suggesting that the herC, gene product is essential for cell growth. The 1832 by E. coli DNA fragment, containing the wild-type allele of the herC, mutation, was cloned and an open reading frame for the HerC protein was determined.     

Genetic analysis of instability in Petunia hybrida

Summary In crossing experiments with Petunia hybrida, new mutations, some unstable, have been found in descendants of plants having an unstable allele of the anthocyanin gene An1. One of the unstable mutations affecting the new anthocyanin gene An11 was genetically analyzed, and it was subsequently established in which step of anthocyanin synthesis that An11 is involved. The discovery of new, unstable mutations at other loci indicates that in Petunia also a relation exists between unstable mutations and the presence of transposable elements in the genome. It was demonstrated that reverted alleles (an1 ^+/+) originating from unstable An1 alleles are less stable than the original wild-type allele An1, and that reversions do not increase the chances of occurrence of new, stable or unstable mutations at other loci. These results provide additional arguments in favour of the hypothesis posed in an earlier paper that reversions of unstable An1 alleles are not the result of excision of the inserted transposable element, but are due to the repair of secondary mutations induced by the insert in the regulatory region of the locus. Consequently, a reverted allele still contains the inserted element that may again induce mutations leading to inactivation of An1.     

Site-selected transposon mutagenesis at the hcf106 locus in maize.

The High chlorophyll fluorescence106 (Hcf106) gene in maize is required for chloroplast membrane biogenesis, and the hcf106-mum1 allele is caused by the insertion of a Robertson's Mutator Mu1 element into the promoter of the gene. Seedlings homozygous for hcf106-mum1 are pale green and die 3 weeks after germination, but only in the presence of Mutator activity conferred by active, autonomous Mu regulatory transposons elsewhere in the genome. When Mutator activity is lost, the mutant phenotype is suppressed, and homozygous plants have an almost wild-type phenotype. To isolate derivative alleles at the hcf106 locus that no longer require Mutator activity for phenotypic expression, we have developed a method for site-selected transposon mutagenesis in maize. This procedure, first described for Caenorhabditis elegans and Drosophila, involves using polymerase chain reaction (PCR) to screen pools of individuals for insertions and deletions in genes of known sequence. Pools of seedlings segregating for the progenitor allele hcf106-mum1 were screened by PCR for insertions and deletions associated with Robertson's Mutator. In a 360-bp target region, two new insertions and one deletion were identified in only 700 Mu-active gametes screened. One of the insertions was in the progenitor hcf106-mum1 allele and the other was in the wild-type allele, but all three new alleles were found to have break-points at the same nucleotide in the first intron. Unlike the hcf-106-mum1 progenitor allele, the deletion and one of the insertions conferred pale green seedling lethal phenotypes in the absence of mutator activity. However, the second insertion had a weak, viable phenotype under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)