Negative crossover interference in maize translocation heterozygotes.

Negative interference describes a situation where two genetic regions have more double crossovers than would be expected considering the crossover rate of each region. We detected negative crossover interference while attempting to genetically map translocation breakpoints in maize. In an attempt to find precedent examples we determined there was negative interference among previously published translocation breakpoint mapping data in maize. It appears that negative interference was greater when the combined map length of the adjacent regions was smaller. Even positive interference appears to have been reduced when the combined lengths of adjacent regions were below 40 cM. Both phenomena can be explained by a reduction in crossovers near the breakpoints or, more specifically, by a failure of regions near breakpoints to become competent for crossovers. A mathematical explanation is provided.

Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1.

Phytic acid (myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate or Ins P(6)) typically represents approximately 75% to 80% of maize (Zea mays) seed total P. Here we describe the origin, inheritance, and seed phenotype of two non-lethal maize low phytic acid mutants, lpa1-1 and lpa2-1. The loci map to two sites on chromosome 1S. Seed phytic acid P is reduced in these mutants by 50% to 66% but seed total P is unaltered. The decrease in phytic acid P in mature lpa1-1 seeds is accompanied by a corresponding increase in inorganic phosphate (P(i)). In mature lpa2-1 seed it is accompanied by increases in P(i) and at least three other myo-inositol (Ins) phosphates (and/or their respective enantiomers): D-Ins(1,2,4,5,6) P(5); D-Ins (1,4,5,6) P(4); and D-Ins(1,2,6) P(3). In both cases the sum of seed P(i) and Ins phosphates (including phytic acid) is constant and similar to that observed in normal seeds. In both mutants P chemistry appears to be perturbed throughout seed development. Homozygosity for either mutant results in a seed dry weight loss, ranging from 4% to 23%. These results indicate that phytic acid metabolism during seed development is not solely responsible for P homeostasis and indicate that the phytic acid concentration typical of a normal maize seed is not essential to seed function.

Analysis of four embryo-specific mutants in Zea mays reveals that incomplete radial organization of the proembryo interferes with subsequent development.

Using confocal laser scanning microscopy we have characterized early and intermediate stages of maize wild-type embryogenesis and compared to mutant development of four different embryo-specific mutations, emb*-8518, emb*-8521, emb*-8537, and emb*-8542. Confocal laser scanning microscopy is well suited to study embryo development in maize in a nondisruptive manner from shortly after fertilization to late stages in embryogenesis. The analysis of the mutant morphology indicated that two of the recessive mutations, emb*-8518 and emb*-8521, cause an early developmental arrest in the proembryo/early transition stage: mutant embryos are unable to enter the morphogenetic phase of embryogenesis. In contrast, homozygous emb*-8537, and emb*-8542 embryos progress at least to the coleoptilar stage and sometimes establish a functional shoot meristem that can determine leaf primordia. The morphological characterization of mutants was confirmed by analysis of the expression pattern of three different marker genes: Lipid transfer protein 2, Zea mays Outer Cell Layer 1, and Knotted 1. Our data indicate that both emb*-8518 and emb*-8521 mutant embryos are impaired in restriction of ZmOCL1 transcripts to the embryonic protoderm and therefore fail to establish a normal radial organization. In contrast, emb*-8537 and emb*-8542 embryos exhibit the wild-type pattern and proceed in development to the formation of a shoot apical meristem and the establishment of bilateral symmetry.

The mac1 mutation alters the developmental fate of the hypodermal cells and their cellular progeny in the maize anther.

In angiosperm ovules and anthers, the hypodermal cell layer provides the progenitors of meiocytes. We have previously reported that the multiple archesporial cells1 (mac1) mutation identifies a gene that plays an important role in the switch of the hypodermal cells from the vegetative pathway to the meiotic (sporogenous) pathway in maize ovules. Here we report that the mac1 mutation alters the developmental fate of the hypodermal cells of the maize anther. In a normal anther a hypodermal cell divides periclinally with the inner cell giving rise to the sporogenous archesporial cells while the outer cell, together with adjacent cells, forms the primary parietal layer. The cells of the parietal layer then undergo two cycles of periclinal divisions to give rise to three wall layers. In mac1 anthers the primary parietal layer usually fails to divide periclinally so that the three wall layers do not form, while the archesporial cells divide excessively and most fail to form microsporocytes. The centrally located mutant microsporocytes are abnormal in appearance and in callose distribution and they fail to proceed through meiosis. These failures in development and function appear to reflect the failure of mac1 gene function in the hypodermal cells and their cellular progeny.

New insights into the role of the maize ameiotic1 locus.

In maize the am1-1 mutant allele results in both the male and female meiocytes undergoing mitosis in place of the meiotic divisions. A second mutant allele am1-praI enables both the male and female meiocytes to proceed to the early zygotene stage of meiotic prophase I before being blocked. Here we report on three new alleles that allow all male meiocytes to undergo mitosis but in female meiocytes approximately one quarter (am1-2), one half (am1-485), or all (am1-489) of them are blocked at an abnormal interphase stage. Previous analysis has shown that am1-praI is dominant to am1-1 in male meiocytes. Cytological analysis of heteroallelic combinations in female meiocytes now indicates a dominance relationship of am1-praI > am1-1 > am1-2/am1-485 > am1-489. The evidence provided by the female phenotypes of the new mutant alleles suggest that, whereas the normal am1 allele is required for the meiocytes to proceed through meiosis, a partially functional allele may be required for their diversion into a mitotic division. The partial or complete blockage of mitosis in female meiocytes carrying the new am1 alleles rules out the possibility that the mitotic division of mutant meiocytes reflects a simple default pathway for cells that cannot initiate meiosis. This locus may have a dual function.

Embryo Sac Development in the Maize indeterminate gametophyte1 Mutant: Abnormal Nuclear Behavior and Defective Microtubule Organization.

The indeterminate gametophyte1 mutation in maize has been known to disrupt development of the female gametophyte. Mutant embryo sacs have abnormal numbers and behavior of micropylar and central cell nuclei, which result in polyembryony and elevated ploidy levels in the endosperm of developing kernels. In this study, we confirm abnormal nuclear behavior and present novel findings. In contrast to the normal form, there is no obvious polarity in two-nucleate embryo sacs or in the micropylar cells of eight-nucleate embryo sacs. We show that the second and third mitoses are not fully synchronized and that additional mitoses can occur in all of the nuclei of the mutant embryo sac or in just the micropylar or central regions. After cellularization, individual micropylar cells can undergo mitosis. Abnormal microtubular behavior results in irregular positioning of the nuclei, asynchronous microtubular patterns in different pairs of nuclei, and abnormal phragmoplasts after the third mitotic division. These results indicate that in addition to acting primarily in controlling nuclear divisions, the indeterminate gametophyte1 gene acts secondarily in regulating microtubule behavior. This cytoskeletal activity most likely controls the polarization and nuclear migration underlying the formation and fate of the cells of the normal embryo sac.

The mac1 gene: controlling the commitment to the meiotic pathway in maize.

The switch from the vegetative to the reproductive pathway of development in flowering plants requires the commitment of the subepidermal cells of the ovules and anthers to enter the meiotic pathway. These cells, the hypodermal cells, either directly or indirectly form the archesporial cells that, in turn, differentiate into the megasporocytes and microsporocytes. We have isolated a recessive pleiotropic mutation that we have termed multiple archesporial cells1 (mac1) and located it to the short arm of chromosome 10. Its cytological phenotype suggests that this locus plays an important role in the switch of the hypodermal cells from the vegetative to the meiotic (sporogenous) pathway in maize ovules. During normal ovule development in maize, only a single hypodermal cell develops into an archesporial cell and this differentiates into the single megasporocyte. In mac1 mutant ovules several hypodermal cells develop into archesporial cells, and the resulting megasporocytes undergo a normal meiosis. More than one megaspore survives in the tetrad and more than one embryo sac is formed in each ovule. Ears on mutant plants show partial sterility resulting from abnormalities in megaspore differentiation and embryo sac formation. The sporophytic expression of this gene is therefore also important for normal female gametophyte development.

Female Gametophyte Development in Maize: Microtubular Organization and Embryo Sac Polarity.

The developmental stages of the maize embryo sac were correlated with the corresponding silk lengths of ear florets in the female inflorescence. The development of embryo sacs in the ovules of spikes occurs in a gradient pattern with the initiation of the embryo sac beginning at the base of the ear and progressing to the top. At the beginning of meiosis, the presence of conspicuous cortical microtubules coincides with the extensive elongation of the megasporocyte. The spindles at metaphase I and II align along the long axis of the megasporocyte leading to the linear alignment of the dyad and tetrad of megaspores. During megagametogenesis, micropylar and chalazal nuclei of the embryo sac undergo synchronized divisions and migration at the second and third mitosis. Radiate perinuclear microtubules are present during the interphase of the second and third mitosis, and inter-sister nuclear microtubules occur at the late four-nucleate embryo sac. The configuration and orientation of the spindles, phragmoplasts, and pairs of nuclei result in precise positioning of the nuclei. The fusion of the polar nuclei and the formation of a microtubule organizing center-like structure in the filiform apparatus occur right after the first division of the antipodal cells. The different patterns of organization of microtubules in the cells of the mature embryo sac reflect their structural adaptations for their future function.

The role of the ameiotic1 gene in the initiation of meiosis and in subsequent meiotic events in maize.

Understanding the initiation of meiosis and the relationship of this event with other key cytogenetic processes are major goals in studying the genetic control of meiosis in higher plants. Our genetic and structural analysis of two mutant alleles of the ameiotic1 gene (am1 and am1-praI) suggest that this locus plays an essential role in the initiation of meiosis in maize. The product of the ameiotic1 gene affects an earlier stage in the meiotic sequence than any other known gene in maize and is important for the irreversible commitment of cells to meiosis and for crucial events marking the passage from premeiotic interphase into prophase I including chromosome synapsis. It appears that the period of ameiotic1 gene function in meiosis at a minimum covers the interval from some point during premeiotic interphase until the early zygotene stage of meiosis. To study the interaction of genes in the progression of meiosis, several double meiotic mutants were constructed. In these double mutants (i) the ameiotic1 mutant allele was brought together with the meiotic mutation (afd1) responsible for the fixation of centromeres in meiosis; and with the mutant alleles of the three meiotic genes that control homologous chromosome segregation (dv1, ms43 and ms28), which impair microtubule organizing center organization, the orientation of the spindle fiber apparatus, and the depolymerization of spindle filaments after the first meiotic division, respectively; (ii) the afd1 mutation was combined with two mutations (dsy1 and as1) affecting homologous pairing; (iii) the ms43 mutation was combined with the as1, the ms28 and the dv1 mutations; and (iv) the ms28 mutation was combined with the dv1 mutation and the ms4 (polymitotic1) mutations. An analysis of gene interaction in the double mutants led us to conclude that the ameiotic1 gene is epistatic over the afd1, the dv1, the ms43 and the ms28 genes but the significance of this relationship requires further analysis. The afd gene appears to function from premeiotic interphase throughout the first meiotic division, but it is likely that its function begins after the start of the ameiotic1 gene expression. The afd1 gene is epistatic over the two synaptic mutations dsy1 and as1 and also over the dv1 mutation. The new ameiotic*-485 and leptotene arrest*-487 mutations isolated from an active Robertson's Mutator stocks take part in the control of the initiation of meiosis.

Isolation and Characterization of 51 embryo-specific Mutations of Maize.

A plant embryo consists of an embryonic axis, which eventually grows into the adult body, and one or two nutritive structures, the cotyledons. In the grasses embryo morphogenesis can be divided into three periods: during the first the embryo is regionalized into an embryo proper and suspensor, during the second the embryonic axis is established, and during the third vegetative structures are elaborated. Maize, with its well-characterized embryo-genesis, powerful genetics, and transposon tagging stocks, offers an attractive system for mutational analysis of these events. We have isolated 51 embryo-specific (emb) mutations from active Robertson's Mutator maize stocks. These are single-gene recessive lethals that represent at least 45 independent mutation events. Each of the 25 mutations was located to a chromosome arm using a B-A translocation set that uncovers approximately 40% of the genome; the same test failed to locate 20 others. The embryo phenotype of 27 mutations was characterized by examining mature mutant embryos in fresh dissection: the various emb mutations differ in phenotype and each is consistent in its expression. All 27 mutations result in retarded embryos that are morphologically abnormal. Nine mutants are blocked during the first period; 10 mutants are blocked during the second period; and eight mutants are blocked during the third period. Based on both the genetic and developmental data, it is likely that there are many loci that can mutate to give the emb phenotype and that these genes are crucial to the morphogenesis of the embryo.

Characterization of the two maize embryo-lethal defective kernel mutants rgh*-1210 and fl*-1253b: effects on embryo and gametophyte development.

We have examined the effects on embryonic and gametophytic development of two nonallelic defective-kernel mutants of maize. Earlier studies indicated that both mutants are abnormal in embryonic morphogenesis as well as in the formation of their endosperm. Mutant rgh*-1210 embryos depart from the normal embryogenic pathway at the proembryo and transition stage, by developing meristematic lobes and losing bilateral symmetry. They continue growth as irregular cell masses that enlarge and become necrotic. Somatic embryos arising in rgh*-1210 callus cultures display the rgh*-1210 mutant phenotype. Mutant fl*-1253B embryos are variably blocked from the coleoptilar stage through stage 2. Following formation of the shoot apex in the mutant embryos the leaf primordia and tissues surrounding the embryonic axis continue growth and cell division, while the scutellum ceases development and becomes hypertrophied. Mutant fl*-1253B embryos are unable to germinate, either in mutant kernels or as immature embryos in culture, and the mutant scutellar tissue does not produce regenerable callus. Expression of the fl*-1253B locus during male gametophytic development is revealed by a marked reduction in pollen transmission as a result of mutant expression during the interval between meiosis and the initiation of pollen tube growth. In both mutants, there is considerable proliferation of the aleurone cells of the endosperm. Mutant expression of rgh*-1210 in the female gametophyte is revealed by the abnormal antipodal cells of the embryo sac. These results show that these two gene loci play unique and crucial roles in normal morphogenesis of the embryo. In addition, it is evident that both mutants are pleiotropic in affecting the development of the endosperm and gametophyte as well as the embryo. These pleiotropisms suggest some commonality in the gene regulation of development in these three tissues.

Viloxazine in the treatment of depressive neurosis: a controlled clinical study with doxepin and placebo.

In a four-week, double-blind, clinical trial thirty-one patients with depressive neurosis were treated with viloxazine, doxepin, or placebo. There were no differences among the three groups in therapeutic effects. Many depressed out-patients improve on placebo. Viloxazine hydrochloride is one of a series of compounds developed to explore the central nervous system activity of the aryloxypropanolamine type of beta-adreno-receptor antagonists. Initial clinical study support the hypothesis that viloxazine has antidepressant properties in man (Bayliss et al, 1974; Bereen, 1973; Pichot et al., 1975; Tsegos and Ekdawi, 1974).

Defective Kernel Mutants of Maize II. Morphological and Embryo Culture Studies.

This report presents the initial results of our study of the immature kernel stage of 150 defective kernel maize mutants. They are single gene, recessive mutants that map throughout the genome, defective in both endosperm and embryo development and, for the most part, lethal (Neuffer and Sheridan 1980). All can be distinguished on immature ears, and 85% of them reveal a mutant phenotype within 11 to 17 days post-pollination. Most have immature kernels that are smaller and lighter in color than their normal counterparts. Forty of the mutants suffer from their defects early in kernel development and are blocked in embryogenesis before their primordia differentiate, or, if primordia are formed, they are unable to germinate when cultured as immature embryos or tested at maturity; a few begin embryo degeneration prior to the time that mutant kernels became visually distinguishable. The others express the associated lesion later in kernel development and form at least one leaf primordium by the time kernels are distinguishable and will germinate when cultured or tested at maturity. In most cases, on a fresh weight basis, the mutants have embryos that are more severely defective than the endosperm; their embryos usually are no more than one-half to two-thirds the size, and lag behind by one or two developmental stages. in comparison with embryos in normal kernels from the same ear. One hundred and two mutants were examined by culturing embryos on basal and enriched media; 21 simply enlarged or completely failed to grow on any of the media tested; and 81 produced shoots and roots on at least one medium. Many grew equally well on basal and enriched media; 16 grew at a faster rate on basal medium and 23 displayed a superior growth on enriched medium. Among the latter group, 10 may be auxotrophs. One of these mutants and another mutant isolated by E. H. Coe are proline-requiring mutants, allelic to pro-1. Considering their diversity of expression as evidenced by their differences in morphological appearance, degree of defectiveness and response to embryo culturing, we believe that they represent many different gene loci.

Defective kernel mutants of maize. I. Genetic and lethality studies.

A planting of 3,919 M(1) kernels from normal ears crossed by EMS-treated pollen produced 3,461 M(1) plants and 3,172 selfed ears. These plants yielded 2,477 (72%) total heritable changes; the selfed ears yielded 2,457 (78%) recessive mutants, including 855 (27%) recessive kernel mutants and 8 (0.23%) viable dominant mutants. The ratio of recessive to dominant mutants was 201:1. The average mutation frequency for four known loci was three per 3,172 genomes analyzed. The estimated total number of loci mutated was 535 and the estimated number of kernel mutant loci mutated was 285. Among the 855 kernel mutants, 432 had a nonviable embryo, and 59 germinated but had a lethal seedling. A sample of 194 of the latter two types was tested for heritability, lethality, chromosome arm location and endosperm-embryo interaction between mutant and nonmutant tissues in special hyper-hypoploid combinations produced by manipulation of B-A translocations. The selected 194 mutants were characterized and catalogued according to endosperm phenotype and investigated to determine their effects on the morphology and development of the associated embryo. The possibility of rescuing some of the lethal mutants by covering the mutant embryo with a normal endosperm was investigated. Ninety of these 194 mutants were located on 17 of the 18 chromosome arms tested. Nineteen of the located mutants were examined to determine the effect of having a normal embryo in the same kernel with a mutant endosperm, and vice versa, as compared to the expression observed in kernels with both embryo and endosperm in a mutant condition. In the first situation, for three of the 19 mutants, the mutant endosperm was less extreme (the embryo helped); for seven cases, the mutant endosperm was more extreme (the embryo hindered); and for nine cases, there was no change. In the reverse situation, for four cases the normal endosperm helped the mutant embryo; for 14 cases there was no change and one case was inconclusive.

Cytochemical and ultrastructural studies on the synaptonemal complex of rat spermatocytes.

The effects of several dehydration treatments on the synaptonemal complex (SC), histone solubility in 2.0 M NaCl, and histone-DNA interaction in unfixed rat spermatocytes were evaluated. Freeze substitution with ethanol or dehydration with polyethylene glygol resulted in loss of the SC, preservation of histone solubility and DNA-histone salt linkages. Dehydration with ethylene gylcol or hexylene glycol resulted in preservation of SC with a clear delineation of attachment of the chromatin fibrils to the lateral elements, but a loss of histone solubility and histone-DNA linkages. Dehydration to a fifty percent concentration with glycerol with completion of dehydration with ethylene glycol had the same effect but also resulted in an even distribution of chromatin fibrils. Dehydration with glycerol alone resulted in clumping of chromatin and loss of SC structure, histone solubility and histone-DNA linkages. Partial dehydration to a fifty percent concentration with these three solvents followed by freeze substitution with ethanol resulted in the loss of SC structure and histone solubility but the preservation of histone-DNA linkages. It is likely that these nonaqueous solvents affected the histone hydrophobic groups and thereby altered histone conformation and interactions. These alterations, depending on the treatment used, resulted in the loss or preservation of SC, histone solubility and histone-DNA interactions thereby indicating that the hydrophobic interactions of the histones are crucial for the preservation of these feature of meiotic chromosomes. These results also demonstrate that neither does the preservation of the histone-DNA salt linkages suffice for the preservation of the SC nor does their disruption necessarily result in its loss. The lysine-rich histones, particularly that one unique to meiotic cells, may through their interactions play a crucial role in SC structure.

Sexual functioning in patients with chronic renal failure.

There is little information on the sexual activity of patients with end-stage renal failure in spite of its being mentioned as a common side effect of kidney disease and chronic hemodialysis. In this report 32 married, male dialysis patients, 19 of whom had also received renal homografts, were interviewed and a detailed sexual history obtained. Twenty per cent of this population had no decrease in sexual functioning after the onset of kidney disease or the institution of dialysis, 45 per cent had reduced sexual potency after the onset of kidney disease, and another 35 per cent after beginning dialysis. Forty per cent of those patients receiving a kidney transplant had a subsequent increase in sexual potency.