SHA1, a novel RING finger protein, functions in shoot apical meristem maintenance in Arabidopsis

Post-embryonic plant growth is dependent on a functional shoot apical meristem (SAM) that provides cells for continuous development of new aerial organs. However, how the SAM is dynamically maintained during vegetative development remains largely unclear. We report here the characterization of a new SAM maintenance mutant, sha1-1 (shoot apical meristem arrest 1-1), that shows a primary SAM-deficient phenotype at the adult stage. The SHA1 gene encodes a novel RING finger protein, and is expressed most intensely in the shoot apex. We show that, in the sha1-1 mutant, the primary SAM develops normally during the juvenile vegetative stage, but cell layer structure becomes disorganized after entering the adult vegetative stage, resulting in a dysfunctional SAM that cannot initiate floral primordia. The sha1-1 SAM terminates completely at the stage when the wild-type begins to bolt, producing adult plants with a primary inflorescence-deficient phenotype. These observations indicate that SHA1, a putative E3 ligase, is required for post-embryonic SAM maintenance by controlling proper cellular organization.     

Mating System and Basidiospore Formation in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

Prototrophic strains recovered from crosses between auxotrophic strains of the lignin-degrading basidiomycete Phanerochaete chrysosporium were induced to fruit. The progeny of most of these self-crosses were prototrophic, indicating that the nuclei of the original prototroph were wild-type recombinants rather than complementary heterokaryons and that the binucleate basidiospores of this organism are homokaryotic. Various wild-type strains were shown to have multinucleate cells lacking clamp connections and to possess a variable number of sterigmata per basidium. Colonies arising from single conidia of various wild-type strains were all capable of producing fruit bodies and basidiospores. In addition, single basidiospores from three wild-type strains all produced fruit bodies and basidiospores. Nonfruiting as well as fruiting isolates were obtained from single basidiospores of five other wild-type strains. Basidiospores from these fruiting isolates always yielded colonies that fruited, again indicating that the spores are homokaryotic. Nonfruiting isolates from the same strain did not produce basidiospores when allowed to form a heterokaryon, implying that these isolates do not represent mating types. All this evidence indicates that P. chrysosporium has a primary homothallic mating system. In addition to fruiting and nonfruiting phenotypes, basidiospores from strain OGC101, a derivative of ME-446, gave rise to colonies which did not grow on cellulose (Cel⁻). The fruiting, nonfruiting, and Cel⁻ phenotypes differed from each other and from the parental wild-type strain in a variety of characteristics, including growth, conidiation, and evolution of ¹⁴CO₂ from ¹⁴C-side chain-labeled lignin, indicating that strain OCG101 is a heterokaryon.     

Generating Autotetraploid Sporophytes and Their Use in Analyzing Mutations Affecting Gametophyte Development in the Fern Ceratopteris

The haploid gametophytes of the fern Ceratopteris richardii are autotrophic and develop independently of the diploid sporophyte plant. While haploid genetics is useful for screening and characterizing mutations affecting gametophyte development in Ceratopteris, it is difficult to assess whether a gametophytic mutation is dominant or recessive or to determine allelism by complementation analysis in a haploid organism. This report describes how apospory can be used to produce genetically marked polyploid sporophytes whose gametophyte progeny are heterozygous for mutations affecting sex determination in the gametophyte and a known recessive mutation affecting the phenotype of both the gametophyte and sporophyte. The segregation ratios of wild-type to mutant phenotypes in the gametophyte progeny of polyploid sporophyte plants indicate that all of the mutations examined are recessive. The presence of many multivalents and few univalents in meiotic chromosome preparations of spore mother cells confirm that the sporophyte plants assayed are polyploid. The DNA content of the sperm of their progeny gametophytes was also found to be approximately twice that of sperm from wild-type haploid gametophytes.     

Gene overexpression: uses, mechanisms, and interpretation

The classical genetic approach for exploring biological pathways typically begins by identifying mutations that cause a phenotype of interest. Overexpression or misexpression of a wild-type gene product, however, can also cause mutant phenotypes, providing geneticists with an alternative yet powerful tool to identify pathway components that might remain undetected using traditional loss-of-function analysis. This review describes the history of overexpression, the mechanisms that are responsible for overexpression phenotypes, tests that begin to distinguish between those mechanisms, the varied ways in which overexpression is used, the methods and reagents available in several organisms, and the relevance of overexpression to human disease.     

Hairless is required for the development of adult sensory organ precursor cells in Drosophila

Reduction of the wild-type activity of the gene Hairless (H) results in two major phenotypic effects on the mechanosensory bristles of adult Drosophila. Bristles are either 'lost' (i.e. the shaft and socket fail to appear) or they exhibit a 'double socket' phenotype, in which the shaft is apparently transformed into a second socket. Analysis of the phenotypes conferred by a series of H mutant genotypes demonstrates (1) that different sensilla exhibit different patterns of response to decreasing levels of H+ function, and (2) that the 'bristle loss' phenotype results from greater loss of H+ function than the 'double socket' phenotype. The systematic study of H allelic combinations enabled us to identify genotypes that reliably produce specific mutant defects in particular positions on the bodies of adult flies. This permitted us to investigate the cellular development of sensilla in these same positions in larvae and pupae and thereby establish the developmental basis for the mutant phenotypes. We have found that H is required for at least two steps of adult sensillum development. In positions where 'double socket' microchaetes appear on the notum of H mutant flies, sensillum precursor cells are present in the developing pupa and divide normally, but their progeny adopt an aberrant spatial arrangement and fail to differentiate correctly. In regions of the notum exhibiting 'bristle loss' in adult H mutants, we were unable at the appropriate stages of development to detect sensillum-specific cell types, the precursor cell divisions that generate them, or the primary precursor cells themselves. Thus, the H 'bristle loss' phenotype appears to reflect a very early defect in sensillum development, namely the failure to specify and/or execute the sensory organ precursor cell fate. This finding indicates that H is one of a small number of identified genes for which the loss-of-function phenotype is the failure of sensillum precursor cell development.     

Mottled coloration in a rainbow trout is associated with mosaicism for albinism

Within a group of rainbow trout at a commercial farm, a single individual was noted for its mottled yellow and dark skin pigmentation. This female fish was reared to sexual maturity and sublots of its eggs were crossed to rainbow trout males from golden, albino, and wild-type (dark-pigmented) strains. Development in other eggs was activated to produce diploid gynogenetic offspring. Coloration within each progeny group was scored at 10 weeks following initiation of feeding. Mottled coloration was observed in none of the progeny at this time. The phenotypes observed (palomino, albino, and/or wild type) and their proportions within progeny groups indicated that the mottled female was originally heterozygous for albinism. The fish apparently became mosaic for this trait following mutation of the wild-type allele at the albinism locus within a cell(s) early in embryonic development. Curiously, at approximately 6 months after initiation of feeding, mottled coloration became apparent in 2 fish from among 25 progeny of the cross to the golden male. No change in phenotype was noted at this time in 9 gynogenetic progeny nor in 68 progeny from the cross to the albino male. Apparently additional mutations and/or other genetic and regulatory processes affecting coloration came into play during juvenile development of these latter two fish.     

YABBY polarity genes mediate the repression of KNOX homeobox genes in Arabidopsis

The YABBY (YAB) genes specify abaxial cell fate in lateral organs in Arabidopsis. Loss-of-function mutants in two early-expressing YAB genes, FILAMENTOUS FLOWER (FIL) and YAB3, do not exhibit vegetative phenotypes as a result of redundancy. Mutations in these genes result in the derepression of the KNOX homeobox genes SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS, and KNAT2 in the leaves and in the partial rescue of stm mutants. Here, we show that fil yab3 double mutants exhibit ectopic meristem formation on the adaxial surfaces of cotyledons and leaf blades. We propose that in addition to abaxial specification, lateral organ development requires YAB function to downregulate KNOTTED homeobox genes so that meristem initiation and growth are restricted to the apex.     

PldB, a putative phospholipase D homologue in Dictyostelium discoideum mediates quorum sensing during development

Quorum sensing, also known as cell-density sensing in the unicellular eukaryote Dictyostelium discoideum, is required for efficient entry into the differentiation and development segment of its life cycle. Quorum sensing is accomplished by simultaneously secreting and sensing the glycoprotein Conditioned Medium Factor, or CMF. When the density of starving cells is high, CMF levels are high, which leads to aggregation followed by development. Here, we describe the role of pldB, a gene coding for a putative phospholipase D (PLD) homologue, in quorum sensing. We find that in submerged culture, adding butanol, an inhibitor of PLD-catalyzed phosphatidic acid production, allows cells to bypass the requirement for CMF mediated quorum sensing and aggregate at low cell density. Deletion of pldB mimics the presence of butanol, allowing cells to aggregate at low cell density. pldB- cells also initiate and finish aggregation rapidly. Analysis of early developmental gene expression in pldB- cells reveals that the cyclic AMP receptor cAR1 is expressed at higher levels earlier than in wild-type cells, which could explain the rapid aggregation phenotype. As would be predicted, cells overexpressing pldB are unable to aggregate even at high cell density. Adding CMF to these pldB- overexpressing cells does not rescue aggregation. Both of these phenotypes are cell autonomous, as mixing a small number of pldB- cells with wild-type cells does not cause the wild-type cells to behave like pldB- cells.     

Short-term rescue by RNA injection of a mitotic arrest mutation that affects the preimplantation mouse embryo

The mutation oligosyndactyly results in syndactyly, abnormal fusion and insertion of certain limb muscles, and diabetes insipidus in heterozygous mice. When homozygous the mutation is lethal; beginning at the blastocyst stage, the homozygous cells arrest in metaphase with intact spindles. The mutant phenotype cannot be corrected by forming aggregation chimeras with wild-type cells, suggesting that the mutation results in a cell autonomous lethal condition. Short-term rescue of the homozygous-induced mitotic arrest can be achieved, however, by cytoplasmic injection of polyadenylated RNA obtained from a rapidly dividing embryo-derived stem cell line.     

Ontogeny of the reproductive shoot apex ofClethra barbinervis,especially on the superficial view

The structure and the ontogenetic process of the reproductive shoot apex forming a terminal inflorescence ofClethra barbinervis were examined, especially concerning the superficial view of the apex. The system of contact parastichies is 2+3 in phyllotaxis in the vegetative phase, changing to 5+8 for bract arrangement in the reproductive phase. At the same time the size of the apex is conspicuously enlarged. The size of the foliage leaf primordia in the vegetative phase is larger than that of the bract primordia in the reproductive phase. The radial cell files, which are clear in the vegetative shoot apex, are not recognizable at least in the early stage of the reproductive phase. The author proposes a close correlation between the appearance of the radial cell files, as well as the construction of the apical sectors, and the sizes of the shoot apex and leaf primordia. It may be proposed also that the construction of the apical sectors is closely correlated with the phyllotaxis.     

Heterochronic mutations affecting shoot development in maize

Three semidominant, nonallelic mutations of maize, Teopod 1 (Tp1), Teopod 2 (Tp2) and Teopod 3 (Tp3), have a profound effect on both vegetative and reproductive development. Although each mutation is phenotypically distinct, they all (1) increase the number of vegetative phytomers; (2) increase the number of phytomers producing ears, tillers and prop roots; (3) increase the number of leaves bearing epidermal wax; (4) decrease the size of leaves and internodes; (5) decrease the size of both the ear and tassel; and (6) transform reproductive structures into vegetative ones. The analysis presented here suggests that this phenotype reflects the prolonged expression of a juvenile, vegetative developmental program which overlaps with the reproductive developmental program. The expression of these mutations is different in each of the four inbred backgrounds used in this study. Tp1 and Tp2 have similar phenotypes and are more highly expressed in the A632 and Oh51a inbred backgrounds than in W23 and Mo17. Tp3 has less extreme effects than either of these mutations and has the opposite modification pattern; i.e., it is more highly expressed in W23 and Mo17 than in A632 and Oh51a. The expression of Tp1 and Tp2 in the presence of varying doses of their wild-type alleles indicate that both are gain-of-function mutations. The phenotypes of Tp1 and Tp2 and the nature of their response to variation in gene dose suggest that they control related, but nonidentical functions. The developmental and evolutionary implications of the heterochronic phenotype of these mutations is discussed.     

Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.     

Isolated fibrils rescue cohesion and development in the Dsp mutant of Myxococcus xanthus.

Extracellular fibrils are involved in cell cohesion and cell development in Myxococcus xanthus. One group of social motility mutants, Dsp, is unable to produce extracellular fibrils; these mutants also lose the abilities to cohere and to develop. Extracellular fibrils isolated from vegetative wild-type cells and added to Dsp cells fully restored the abilities of these cells to cohere and to undergo normal morphological development. The fibrils thus mimic the ability of intact, wild-type cells to carry out the same rescue. Optimal cohesion rescue by fibrils required calcium and magnesium ions, did not require protein synthesis, but was energy dependent, i.e., sodium azide and sodium cyanide blocked rescue. Cohesion rescue was also blocked by the diazo dye Congo red. Cohesion rescue is genus specific, i.e., isolated fibrils did not cause the cohesion of Pseudomonas aeruginosa, Bacillus subtilis, Proteus mirabilis, Escherichia coli, or the related myxobacterium Stigmatella aurantiaca. Developmental rescue of Dsp by isolated fibrils included aggregation, fruiting body formation, and myxospore morphogenesis. Developmental gene expression in the Dsp mutant was only partially rescued by the isolated fibrils.     

Positive Selection for Mating with Functional Heterokaryons in TETRAHYMENA PYRIFORMIS

Allelic assortment of Tetrahymena pyriformis makes possible the isolation of cells which contain both a heterozygous germ line nucleus and a somatic nucleus which expresses phenotypes of only one member of allelic pairs. Cycloheximide-sensitive segregants have been isolated from the vegetative progeny of cells heterozygous for a dominant mutation conferring resistance to cycloheximide; such segregants are called functional heterokaryons. Since progeny from crosses of these cells are cycloheximide-resistant, addition of the drug provides positive selection for successful exconjugants. The timing for expression of the new phenotype during conjugation is presented and used to identify what appear to be immature progeny of round one mating in genomic exclusion. Usefulness of functional heterokaryons in a number of genetic and developmental studies is discussed.     

TIMING AND LIGHT REGULATION OF APICAL MORPHOGENESIS DURING REPRODUCTIVE DEVELOPMENT IN WILD-TYPE POPULATIONS OF ACETABULARIA ACETABULUM (CHLOROPHYCEAE)

In the giant unicellular green alga, Acetabularia acetabulum (L.) Silva, development is altered by light. For example, blue light induces the vegetative apex to produce whorls of hairs that encircle the stalk and, later, blue light may trigger reproductive onset. The two goals of this study were to determine when changes in apical shape occur during formation of the reproductive structure, or "cap," and to determine which of these differentiation events require light. The first visible indication of cap initiation was a rounded swelling of the apex, which we call a knob-shaped apex (time = 0 hours). Subsequent changes in shape were a hyaline, knob-shaped apex, reached by 50% of the population 3 h later, and the formation of a whorl of unilobed chambers at 16 h. These chambers became bilobed at 33 h and trilobed at 34 h. Successive sets of cap hairs grew from protuberances found on the surface of the uppermost lobes of the chambers (superior corona). After knob, the remainder of cap formation was largely independent of light. However, the initiation of each set of cap hairs required light. If a recently initiated cap was amputated, the individual recapitulated development, repeating a portion of vegetative morphogenesis (i.e. it made whorls of sterile hairs) before initiating a new cap. The developmental sequence between amputation and initiation of a new cap required light. A model for light-regulated changes in shape at the apex of Acetabularia acetabulum, which integrates whorl and cap formation and encompasses both vegetative and reproductive development of this organism, is presented.     

Gene dosage and selective expression modify phenotype in a Drosophila model of human mitochondrial disease

Human mitochondrial disease manifests with a wide range of clinical phenotypes of varying severity. To create a model for these disorders, we have manipulated the Drosophila gene technical knockout, encoding mitoribosomal protein S12. Various permutations of endogenous and transgenic alleles create a range of phenotypes, varying from larval developmental arrest through to mild neurological defects in the adult, and also mimic threshold effects associated with human mtDNA disease. Nuclear genetic background influences mutant phenotype by a compensatory mechanism affecting mitochondrial RNA levels. Selective expression of the wild-type allele indicates critical times and cell-types in development, in which mitochondrial protein synthesis deficiency leads to specific phenotypic outcomes.     

Hypervariability, a new phenomenon of genetic instability, related to DNA amplification in Streptomyces ambofaciens.

The wild-type strain Streptomyces ambofaciens DSM 40697 exhibits a high degree of genetic instability. Pigment-defective colonies were observed in the progeny of wild-type colonies at a frequency of about 0.01. While only 13% of these pigment-defective colonies gave rise to homogeneous progeny exhibiting the mutant parental phenotype, 87% of the mutant colonies gave rise to hetergeneous progeny without a preponderant phenotype. This new phenomenon of instability was called hypervariability. In addition, 21% of the mutant strains arising in hypervariable progeny contained highly reiterated DNA sequences, while amplified DNA sequences could be detected in neither stable pigment-defective mutant clones nor in wild-type clones. These results indicate a frequent association between genetic instability and hypervariability and a frequent association between hypervariability and amplification of DNA sequences.