Region-specific cis- and trans-acting factors contribute to genetic variability in meiotic recombination in maize.

Understanding the genetic basis for variability in recombination rates is important for general genetic studies and plant-breeding efforts. Earlier studies had suggested increased recombination frequencies in particular F2 populations derived from the maize inbred A188. A detailed phenotypic and molecular analysis was undertaken to extend these observations and dissect the responsible factors. A heritable increase in recombination in the sh1-bz1 interval was observed in these populations. A factor causing an approximate twofold increase mapped to the A188 sh1-Bz1 region, behaved as a dominant, cis-acting factor, affected recombination equally in male and female sporogenesis and did not reduce the well-studied complete interference in the adjacent bz1-wx interval. This factor also did not increase recombination frequencies in the c1-sh1 and bz1-wx intervals, demonstrating independent control of recombination in adjacent intervals. Additional phenotypic analysis of recombination in the c1-sh1 and bz1-wx intervals and RFLP analysis of recombination along chromosomes 7 and 5 suggested that heritable factors controlling recombination in these intervals act largely independently and in trans. Our results show that recombination in these populations, and possibly maize in general, is controlled by both cis- and trans-acting factors that affect specific chromosomal regions.

Characterization of a meiotic crossover in maize identified by a restriction fragment length polymorphism-based method.

Genetic map lengths do not correlate directly with genome size, suggesting that meiotic recombination is not uniform throughout the genome. Further, the abundance of repeated sequences in plant genomes requires that crossing over is restricted to particular genomic regions. We used a physical mapping approach to identify these regions without the bias introduced by phenotypic selection. This approach is based on the detection of nonparental polymorphisms formed by recombination between polymorphic alleles. In an F2 population of 48 maize plants, we identified a crossover at two of the seven restriction fragment length polymorphism loci tested. Characterization of one recombination event revealed that the crossover mapped within a 534-bp region of perfect homology between the parental alleles embedded in a 2773-bp unique sequence. No transcripts from this region could be detected. Sequences immediately surrounding the crossover site were not detectably methylated, except for an SstI site and at the flanking repetitive sequences were faithfully inherited by the recombinant allele. Our observations suggest that meiotic recombination in maize occurs between perfectly homologous sequences, within unmethylated, nonrepetitive regions of the genome.

Zeon-1, a member of a new maize retrotransposon family.

We have previously shown that the tandemly duplicated 27 kDa maize storage protein locus underwent mitotic rearrangement to yield a single-copy allele in isolates of the inbred line A188. This rearrangement contains a new LTR retrotransposon, designated Zeon-1. This middle repetitive element of 7313 bp had two long terminal repeats, a primer binding site, a polypurine tract and a gag-related open reading frame of 375 amino acids. Transcripts of the gag-related region were detected by the polymerase chain reaction (PCR) in certain maize tissues, and Western blots detected the gag-related protein in the same tissues. Moreover, the product of this mitotic rearrangement was shown to contain the same insertion site and 3' LTR as Zeon-1, suggesting that this rearrangement occurs with unusual precision. Zeon elements were found to be present in teosinte and not present in the Gramineae wheat, barley, sorghum and rye.

Variegated phenotype and developmental methylation changes of a maize allele originating from epimutation.

Two instances of genetic transmission of spontaneous epimutation of the maize P-rr gene were identified. Transmission gave rise to two similar, moderately stable alleles, designated P-pr-1 and P-pr-2, that exhibited Mendelian behavior. Both isolates of P-pr conditioned a variable and variegated phenotype, unlike the uniform pigmentation conditioned by P-rr. Extensive genomic analysis failed to reveal insertions, deletions or restriction site polymorphisms between the new allele and its progenitor. However, methylation of the P gene was increased in P-pr relative to P-rr, and was greatly reduced (though not lost) in a revertant to uniform pigmentation. Variability in pigmentation conditioned by P-pr correlated with variability in transcript levels of the P gene, and both correlated inversely with variability in its methylation. Part of the variability in methylation could be accounted for by a developmental decrease in methylation in all tissues of plants carrying P-pr. We hypothesize that the variegated phenotype results from a general epigenetic pathway which causes a progressive decrease in methylation and increase in expression potential of the P gene as a function of cell divisions in each meristem of the plant. This renders all tissues chimeric for a functional gene; chimerism is visualized as variegation only in pericarp due to the tissue specificity of P gene expression. Therefore, this allele that originates from epimutation may exemplify an epigenetic mechanism for variegation in maize.

Trans replication and high copy numbers of wheat dwarf virus vectors in maize cells.

The replication of shuttle vectors derived from Wheat Dwarf Virus, a monopartite geminivirus, was studied in cultured maize endosperm cells, and in the Black Mexican Sweet (BMS) maize cell line. Using in vivo labeling and DNA methylation analysis, we showed that replication was initiated within 24 hrs after transfection, and did not require cell division in both cell lines. Copy numbers of 30,000 ds DNA molecules per cell were observed in endosperm cells after three days. The replication protein was shown to act in trans, since the wild type gene of the shuttle vector enabled replication-deficient vectors carrying mutated genes to replicate. These properties suggest that WDV may have similar applications in plants as SV40 in mammalian cells.

Cytological aberrations in maize populations exhibiting unusual recombinational behaviour.

Earlier studies identified higher recombinational activity and increased somatic instability at various loci in the F1 and F2 progeny of particular stocks of a maize inbred line, A188. Hypothesizing that this may reflect a genome-wide perturbation of chromosome structure which would be detectable cytologically, aberrant meiotic behaviour in these stocks was investigated. Microsporocytes from a population of F2 plants derived from these stocks displayed the following aberrations: varying frequencies of metaphase and anaphase laggards, 'stickiness' at anaphase I resulting in chromosome bridges from pole to pole, acentric fragments and a spontaneous translocation of the NOR on chromosome 6. The frequencies of metaphase I and anaphase I abnormalities in these stocks were higher to a statistically significant extent than in control F2 populations, suggesting that these aberrations may be related to the unusual genetic behaviour of these stocks.

Sequence variation between alleles reveals two types of copy correction at the 27-kDa zein locus of maize.

In many inbred lines of maize, two 27-kDa storage protein (zein) genes are found within tandem duplications of 12 kb. Both genes of the duplicated allele from the maize inbred line A188 were sequenced and compared to a similar duplicated allele in another inbred line, W22, and to a single-copy allele in the inbred line W64A. The comparisons reveal interesting patterns in the distribution of sequence changes between these alleles. Differences between the two duplicated alleles that are conserved between the two genes of each allele are found exclusively in the 5' region. In contrast, differences between the individual genes of each allele in the 3' region are conserved between the two alleles. The first case is indicative of an intraallelic copy correction mechanism, whereas the second may result from interallelic copy correction. These may be mediated by gene conversion processes, as previously described for other multigene families.

A new allele of the duplicated 27kD zein locus of maize generated by homologous recombination.

The 27kD zein storage protein locus in many inbred lines of maize consists of a tandem duplication of 12kb, with an expressed gene in each repeat, termed A and B. A single-copy allele with only the A gene can be generated from this duplication in particular stocks of the maize inbred line A188 by a mitotic process that includes a crossover at the 3' regions of the two genes (1). Here, we characterize a second single-copy allele with only the B gene, found in different stocks of A188. This allele arises from a homologous recombination at the highly conserved 5' regions of the two repeats, and cloning and sequencing of this allele define the crossover region. The A and B genes in the duplicated allele were previously shown to be expressed at different levels; this difference remains unchanged in either recombinant allele. Therefore, the crossover points of these two recombinant alleles define the borders of cis-acting sequences responsible for the differential expression of the two genes.

A somatic gene rearrangement contributing to genetic diversity in maize.

We have discovered a somatic genomic rearrangement that occurs at high frequency at a duplicated zein locus in certain cultures of the maize inbred line A188. The rearranged allele arises from the duplication by a two-step process involving a homologous recombination and a second event, which may be a deletion, inversion, or insertion; both steps always occur together. The frequency of rearrangement is lower in homozygous states of the parental allele than in heterozygotes. In both cases, the rearrangement is shown to be mitotic. The rearranged product can be transmitted through meiosis, providing another mechanism for genome evolution in higher eukaryotes.

Allelic variation and differential expression at the 27-kilodalton zein locus in maize.

Allelic variation between inbred lines at the 27-kilodalton zein gene locus in maize has been used to study gene expression in developing endosperm. The inbred lines W22 and W23 contain two genes for this protein within two tandem repeats; the individual genes are virtually identical, with 99.9% homology in the 5'-flanking regions. Using gene-specific oligonucleotide probes, we have shown that transcripts of the downstream gene are found at a 2.5-fold-higher level than those of the upstream gene. Another inbred line, BSSS53, has one copy of the gene which is a recombinant of the duplicated genes at the 3'-flanking region. This line has been used in reciprocal crosses to demonstrate dosage effects for the overexpression of the downstream gene and to show that the overexpression of mRNA is reflected in a corresponding increase in the protein level. The accumulation of the protein through development does not, however, always correspond to the difference in mRNA levels.

Fluorographic detection of tritiated glycopeptides and oligosaccharides separated on polyacrylamide gels: analysis of glycans from Dictyostelium discoideum glycoproteins.

Previous workers have shown that oligosaccharides and glycopeptides can be separated by electrophoresis in buffers containing borate ions. However, normal fluorography of tritium-labeled structures cannot be performed because the glycans are soluble and can diffuse during equilibration with scintillants. This problem has been circumvented by equilibration of the gel with 2,5-diphenyloxazole (PPO) prior to electrophoresis. The presence of PPO in the gel during electrophoresis does not alter mobility of the glycopeptides and oligosaccharides. After electrophoresis, the gel is simply dried and fluorography performed. This allows sensitive and precise comparisons of labeled samples in parallel lanes of a slab gel and, since mobilities are highly reproducible, between different gels. The procedure is preparative in that after fluorography the gel bands can be quantitatively eluted for further study, without any apparent modification by the procedure. In this report, the procedure is illustrated by fractionation of both neutral and anionic glycopeptides produced by the cellular slime mold Dictyostelium discoideum.

Glycoprotein biosynthesis in dictyostelium discoideum: developmental regulation of the protein-linked glycans.

The biosynthesis of glycoprotein N-linked oligosaccharides in D. discoideum is initiated by the transfer of a large precursor glycan from a carrier lipid. The subsequent processing of this precursor is dramatically dependent upon the stage of development. In early development processing retains the high mannose structure of the precursor and modifies some glycans by addition of fucose to core sugars and sulfate and phosphate to others. These reactions are coordinately lost during aggregation. Processing in late development extensively trims the precursor and adds fucose to peripheral mannose units of the smallest glycans. These reactions appear coincident with formation of tips on cell mounds. Experiments in which cells were starved in shaken suspension suggest that intercellular contacts and cyclic AMP signals may be sufficient to cause the controlled expression of these two alternate sets of processing enzymes.

Guanidine hydrochloride-induced shedding of a Dictyostelium discoideum plasma membrane fraction enriched in the cyclic adenosine 3',5'-monophosphate receptor.

The cell surface cyclic AMP receptor of Dictyostelium discoideum is under study in a number of laboratories with respect to both its role in development of the organism and the physiology of excitation-response coupling. We report here that when starved amoebae are exposed to the chaotrope guanidine hydrochloride at 1.8 M, they shed a particulate cyclic AMP binding activity into the medium. This activity is due to membrane vesicles which originate from the cell surface. The vesicles are enriched up to 150-fold in cyclic AMP binding activity and up to 14-fold in phospholipid content when compared to the starting amoebae. The cyclic AMP binding activity of the membrane vesicles is identical to that of the cell surface receptor with respect to the following properties; (i) it is lacking in preparations from unstarved, vegetative amoebae; (ii) it is not inhibited by cyclic GMP and is stimulated by calcium ions; (iii) it has very rapid rates of association and dissociation of bound cyclic AMP; (iv) it has two classes of binding sites with dissociation constants similar to those of the surface receptors of whole amoebae. The binding activity of the isolated membranes is stable for several days at 4 degrees C and the lower affinity binding sites are stable up to several months when stored at -80 degrees C. Due to enrichment and stability of the receptor in this preparation, it should be highly suitable for many types of studies. The usefulness is enhanced by the fact that the preparation does not contain detectable cyclic AMP phosphodiesterase activity.

A new radioactive cross-linking reagent for studying the interactions of proteins.

We have developed a photoactivable, heterobifunctional, reversible, radioactively labeled, chemical cross-linking reagent, 3-[(2-nitro-4-azidophenyl)-2-aminoethyldithio]-N-succinimidyl propionate, for studying the interactions of proteins in situ. When reacted in the dark with a purified protein, it forms a covalent derivative which can be purified and reconstituted into biological systems. This derivative will form cross-links to neighboring macromolecules only upon photolysis; reduction cleaves the cross-link and transfers the radiolabel to the second molecule, which can then be identified by standard techniques. We have tested the cross-linker using the binding of gelatin to fibronectin. The cross-linker gives the proper chemical behavior under biological conditions, reacts with high yield and with a very low level of nonspecific cross-linking, and can be used to identify protein-protein and other interactions at the cell surface and elsewhere. The advantages, limitations and possible uses for this reagent are discussed.

Developmental regulation of glycoprotein biosynthesis in Dictyostelium.

We have examined the glycoprotein-linked oligosaccharides assembled during the life cycle of Dictyostelium discoideum, and found their expression to be dramatically dependent upon the stage of development. During early development mature glycans have a high mannose character, and a substantial proportion acquire a fucose residue that correlates with endo-H resistance. One-third of the glycans also acquire sulfate residues. These glycans diminish in importance during aggregation. The mature glycans expressed during aggregation. The mature glycans expressed during late development contain fewer mannose residues, from five to ten mannose residues, and are characterized by the absence of sulfate residues and by the presence of fucose residues on endo-H-sensitive glycans. These glycans make their appearance coincident with the construction of tips on tight cell mounds. At this stage glycans characteristic of both early and late stages occur simultaneously. Developmental regulation of the wide array of protein-linked glycans expressed during the life cycle of Dictyostelium discoideum may be as simple as the controlled transition from a group of structures that are assembled by the vegetative cells to a group of structures that are assembled by the terminally differentiating cells. The potential biological significance of this transition is discussed.