Novel peroxisome clustering mutants and peroxisome biogenesis mutants of Saccharomyces cerevisiae

The goal of this research is to identify and characterize the protein machinery that functions in the intracellular translocation and assembly of peroxisomal proteins in Saccharomyces cerevisiae. Several genes encoding proteins that are essential for this process have been identified previously by Kunau and collaborators, but the mutant collection was incomplete. We have devised a positive selection procedure that identifies new mutants lacking peroxisomes or peroxisomal function. Immunofluorescence procedures for yeast were simplified so that these mutants could be rapidly and efficiently screened for those in which peroxisome biogenesis is impaired. With these tools, we have identified four complementation groups of peroxisome biogenesis mutants, and one group that appears to express reduced amounts of peroxisomal proteins. Two of our mutants lack recognizable peroxisomes, although they might contain peroxisomal membrane ghosts like those found in Zellweger syndrome. Two are selectively defective in packaging peroxisomal proteins and moreover show striking intracellular clustering of the peroxisomes. The distribution of mutants among complementation groups implies that the collection of peroxisome biogenesis mutants is still incomplete. With the procedures described, it should prove straightforward to isolate mutants from additional complementation groups.     

Gibberellin mutants

Research on gibberellin (GA) mutants is reviewed, focusing on reports, published since 1993. The mutants have usually been identified via a shoot elongation screen. This screen exposes mutations influencing GA synthesis, deactivation and reception, and also those acting further down the elongation pathway. Mutations blocking synthesis lead to a dwarf. GA-responsive phenotype. Numerous such mutations are now known. For some steps homologous mutations are known across 4 to 6 model species. Examples include the early step, geranylgeranyl diphosphate to copalyl diphosphate, and the activation step, GA₂₆to GA₁. Several GA-synthesis mutations have now been characterised at the molecular level and all are in structural genes. It is now clear some steps are controlled by gene families with distinct tissue specificity. Further, some enzymes control more than one step in the biosynthetic pathway. The only mutation known to block deactivation. sin in pea, leads to an elongated phenotype. The GA response mutants are less well understood and are a more diverse group. They include elongated mutants with a constitutive GA response (spy in arabidopsis. la cry-s in pea and sln in barley) or an enhanced GA response (phyB in arabidopsis. lv in pea and Ih in cucumber). Short response mutants include at least three types. One group accumulates GAs and are mostly unresponsive to applied GA (gai in arabidopsis. D8 in maize. Rht3 in wheat). A recently identified group, exemplified by Igr in pea and gas in barley, have a short stature and reduced response but attain full responses with very high doses of exogenous GA. How close these mutations act to GA reception remains to be determined. Lastly, a number of mutants with short stature and reduced GA response differ in overall phenotype from GA-deficient plants and cannot be made to mimic wild type even at high GA application rates. These mutations act beyond GA reception and some have already proved useful in elucidating other pathways that affect shoot elongation. For example, the lk and lkb mutations in pea appear to block brassinolide synthesis and this in turn prevents normal GA-mediated elongation responses.     

Plastome mutants

Summary Since the first reports seventy-five years ago on the non-Mendelian inheritance of variegation in plants, chloroplast gene mutations have been useful for genetical and physiological investigations. The mutations have been shown to affect the chloroplast translational apparatus, photosystem I, photosystem II, the cytochrome f/b₆ complex, carbon fixation, or the ATP synthase. They arose spontaneously or were induced by mutagens or by the action of nuclear mutator genes. Alterations of chloroplast DNA include point mutations, deletions, duplications, and inversions. In 1909, Correns discovered uniparental transmission of chloroplasts when he observed the maternal inheritance of a chlorophyll deficiency inMirabilis jalapa. At the same time, Baur (1909) reported crosses ofPelargonium zonale in which the offspring inherited chloroplasts from both parents (biparental transmission) with variegated leaves resulting as the green and white plastids sorted out. since the experiments of Baur and Correns, many non-Mendelian mutants have been isolated in both higher plants and algae (for reviews see Hagemann, 1964; Kirk and Tilney-Bassett, 1978; Gillham, 1978). Some of these are mitochondrial traits, including cytoplasmic male sterility in maize and several other plants (Hanson and Conde, 1985; Pring and Lonsdale, 1985). Several other traits have been tentatively identified as mitochondrial since their inheritance pattern differs from that of both nuclear and chloroplast genes, including the deformed leaf (“falsifolia”) syndrome ofOenothera (Stubbe, 1970), non-chromosomal stripe of maize (Coe, 1983), and inChlamydomonas, photoautotropism (Wiseman et al., 1977) and a minute colony phenotype (Alexander et al., 1974). A far larger number of extranuclear mutations affect the plastome (plastid genome). Among the algae,Euglena gracilis (Russell and Lyman, 1982),Scenedesmus obliquus (Bishop, 1982) andChlorella (Galling, 1982) have yielded interesting mutants, but unlikeChlamydomonas, they are not known to undergo sexual reproduction, and thus the Mendelian or non-Mendelian nature of the mutations has not been determined. Most of the plastome mutations which have been characterized have been isolated in higher plant lines or fromChlamydomonas.     

Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice

In rice, many dwarf mutants have been isolated and characterized. We have investigated the relationship between dwarfism and the gibberellin (GA)-mediated control of physiological processes. Twenty-three rice cultivars and mutants (9 normal, 3 semi-dwarf, 11 dwarf) were analyzed in terms of two GA-mediated processes, namely, elongation of shoots and production of -amylase activity in the endosperm. As a result, we identified four different groups (groups N, T, D and E). Two-dimensional plotting of the extent of induction of -amylase in the endosperm versus the extent of enhancement of shoot elongation upon treatment with exogenous gibberellic acid (GA₃) provided a useful method for the rapid allocation of large numbers of dwarf mutants of rice to the various groups. Members of group N (normal type), which included all normal cultivars and semi-dwarf mutants, showed a slight increase in elongation of shoots and a remarkable increase in production of -amylase with the application of GA₃ during germination. All of the dwarf mutants were classified as being members of the other three groups. Members of group T (Tan-ginbozu type), including three dwarf mutants, were highly responsive to exogenous GA₃ in terms of elongation of shoots and production of -amylase, with associated lower levels of endogenous GA. In contrast, members of the other three groups, including group N, had normal levels of endogenous GAs. Members of group D (Daikoku type) were only slightly responsive to exogenous GA₃, an indication that they are GA-insensitive mutants. Members of group E (Ebisu type) had responses to GA₃ similar to those of group N, not only in terms of elongation of shoots but also in terms of -amylase production, an indication that they are dwarf mutants that can be considered as neither GA-deficient nor GA-insensitive mutants. We also examined a GA-insensitive mutant selected from among 19 near-isogenic dwarf lines of Shiokari, and we concluded that the d-1 gene is associated with the phenotype of GA-insensitive dwarf mutants.     

Plant hormone mutants

Many of the basic facts about plant hormones are still obscure, including biosynthetic pathways and their regulation. Furthermore, our knowledge of the molecular steps between hormones and their action is extremely limited. The increasing collection of isogenic genotypes differing in hormone synthesis or responses offers great promise for future research.     

Cell wall mutants

An ever growing collection of cell wall mutants is yielding new insights into the mechanisms underlying the synthesis and assembly of cell walls in plants. In this review, we will provide an update on the use of genetic tools in plant cell wall research and we will discuss the lessons that can be drawn from the study of the first generation of mutants.     

Phenocopies ofBithorax mutants

Summary Exposure to ether of wild-type embryos of different strains ofDrosophila melanogaster causes phenocopies of different alleles of thebithorax system. Clonal analysis of the phenocopy spots has shown that the transformation caused by the treatment is maintained by cell heredity. Embryos heterozygous for several recessive mutant alleles ofbithorax show the same frequency of phenocopies as wild-type homozygous sib controls. The same holds for embryos heterozygous for the dominant mutant allelesCbx andUbx ¹ which are point mutants in thecis-regulatory region of the system. However, for dominant mutants which have breakpoints in this region (Ubx ⁸⁰,Ubx ¹³⁰ andHm) the frequency of phenocopies is about twice that of their sib controls. Embryos with increasing numbers of copies (from 1 to 4) of thebithorax system show a decreasing frequency of phenocopies. A model is proposed that explains bithorax phenocopies as resulting from disturbances in the distribution of positional information signals for segments (inductor molecules) which compete with the product of a regulator gene (repressor) and thecis-regulatory region of thebithorax system. On this model, the initiation of a metathoracic developmental pathway would result from the derepression of thebithorax system.     

Plant hormone mutants

The techniques used for the production and identification of plant hormone mutants are described. The properties used to classify these mutants into the broad synthesis and response categories are discussed, and the genetic considerations needed to allow their effective use in plant hormone research examined. A brief outline of significant work on gibberellin (GA), abscisic acid (ABA), auxin, ethylene, cytokinin and phytochrome mutants is provided. The molecular action of these genes is discussed where available and recent rapid advances made in Arabidopsis highlighted. Suggestions for future emphasis are made, particularly relating to an examination of the tissue and ontogenetic specificity of the plant hormone genes.     

Phytochrome chromophore-deficient mutants

Phytochrome chromophore-deficient mutants have been used as phytochrome-deficient plants to study many aspects of plant development. However, there are still a number of important questions to be resolved concerning both the targets and the phenotypic consequences of these mutations. Recently, progress has been made in our understanding of the molecular basis of the chromophore deficiency in these mutants. Biochemical assays for the committed steps of chromophore synthesis have been developed and used to demonstrate that the pcd1 and yellow-green-2 mutants of pea and tomato, respectively, are unable to synthesize biliverdin IXα from heme while pcd2 and aurea are deficient in phytochromobilin synthase activity. This review focuses on how this information can be used to help understand the basis of other chromophore-deficient mutants, such as the hy1 and hy2 mutants of Arabidopsis, and discusses how the phenotype of chromophore-deficient mutants is related to lesions in the chromophore biosynthesis pathway.     

Saving zebrafish mutants

At present, the zebrafish Danio rerio is the only vertebrate species for which a large-scale mutagenesis effort to identify developmental genes has been reported. Several laboratories are now intensely pursuing the molecular characterization of the genes affected by these mutations. One important criterion for the identity of the mutated gene is the rescue of the mutant phenotype by a wild-type (wt) copy of the gene. Until recently, most rescue attempts were carried out by injecting wt messenger RNA (mRNA) into fertilized eggs. A report by Yan and collaborators shows the partial rescue of floatinghead mutants by injection of genomic fragments cloned in either bacterial artificial chromosomes or bacteriophage lambda vectors. Combined with other ongoing efforts to characterize the zebrafish genome, this approach of mutant rescue opens interesting avenues for a systematic functional analysis of vertebrate genes.     

The axolotl mutants

The Mexican axolotl (Ambystoma mexicanum) has enjoyed wide use in experimental embryology for over 100 yr. Its usefulness has been extended into the area of developmental genetics largely due to the contributions of R. Briggs and R. R. Humphrey at Indiana University. To date over 30 mutants have been described, almost all of which affect development. Some of these have been discovered in inbred strains while others have been uncovered in recent Mexican imports. These mutants can be subdivided into several major classes. Maternal effect mutations lead to deficiencies in informational, structural, or metabolic components of the egg essential to early development prior to the time at which the embryo's own genome becomes active. In contrast, the developmental lethals affect later stages in embryogenesis when both morphogenetic and biochemical events are determined exclusively by the genotype of the embryo. Most lead to death at about feeding stage. Some, the cell lethals, are believed to suffer from fundamental metabolic defects affecting all parts of the embryo. Others affect the development of specific organs or tissues. The developmental nonlethals also affect specific systems, but ones that are not essential to survival. Some affect the development and survival of pigment cells and these, along with isozyme variants, are useful as markers in developmental experiments. A number of the mutants have been studied in detail, but others scarcely at all. The purpose of this review is to bring them to the attention of all developmental biologists in the hope that their potential will be even more widely recognized.     

Plasmodium berghei: in vivo generation and selection of karyotype mutants and non-gametocyte producer mutants.

We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in vivo asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in vivo. Gametocyte production and karyotype pattern were established at regular intervals during prolonged periods of asexual multiplication of clone 8417 of P. berghei. We found that karyotype mutants and mutants which do not produce gametocytes can replace the original high-producer parasites of clone 8417 within several weeks. The time at which mutants became predominant in the population in different experiments, however, differed greatly. Mutants with intermediate or low gametocyte production were not found. In experimentally mixed infections, containing parasites from two clones from different strains (clone 8417 of the ANKA strain; clone 1 of the K173 strain), high-producer parasites of clone 8417 were overgrown by parasites of the nonproducer clone. Nonproducer mutants from the originally high-producer clone 8417, however, were able to coexist with parasites of the nonproducer clone. These results demonstrate that in our experiments nonproducer parasites had a strong selective advantage during asexual multiplication compared to high producers. All karyotype mutants which became predominant in our experiments were nonproducers. In two experiments a change in karyotype coincided with the loss of gametocyte production which may suggest a causal relationship between these events.