Genetic control of somatic cell differentiation in Volvox analysis of somatic regenerator mutants

The somatic regenerator (reg) mutants of Volvox carteri affect the ability of the normally terminally differentiated somatic cells to establish and/or maintain the differentiated state. Thirty-nine reg mutants of four phenotypic classes have been mapped to two, unlinked genes, regA and regB. Mutants at the regA locus have one of three phenotypes: All somatic cells regenerate new spheroids, somatic cells in the spheroid posterior region regenerate while those in the anterior region differentiate as somatic cells, or regenerating and nonregenerating cells are randomly intermixed. The regB mutant has a random intermixture of regenerating and nonregenerating cells. Somatic cells regenerate new Volvox spheroids in two ways; the cells lose their characteristic shape, become immotile, enlarge and undergo cleavage similar to that of normal reproductive cells or undergo cell division without prior enlargement or loss of cell shape. Temperature shift experiments on a cold-sensitive reg mutant suggest that the gene product acts after the somatic cell initials are formed at the end of cleavage.     

Spontaneous frequency of a developmental mutant in Volvox

Two types of mutants, those resistant to the base analog 5-bromo-2′-deoxyuridine (BrdU) and somatic regenerator (SR) mutants, have been analyzed in Volvox carteri. In somatic regenerator mutants, the somatic cells which are normally terminally differentiated dedifferentiate and regenerate gonidia [Sessoms, A., and Huskey, R. J. (1973). Proc. Nat. Acad. Sci. USA70, 1335–1338; Starr, R. C. (1970). Develop. Biol. Suppl.4, 59–100]. The SR phenotype allows recovery of SR mutations arising in somatic cells, since such somatic cells would regenerate gonidia and give rise to mutant clones. Mutants of any phenotype other than SR can only be recovered if the mutation first appears in a gonidium. Since the somatic cells are 100-fold more numerous than reproductive cells (gonidia), we have determined the spontaneous frequency of both somatic regenerator mutants and mutations to BrdU resistance in order to determine if the SR mutation exerts its effect in the gonidium or in the somatic cell. The two frequencies were found to be nearly identical, suggesting that the SR mutation must first appear in a gonidium in order to be expressed.     

Molecular Analysis of Diepoxybutane-Induced Mutations at the rosy Locus of Drosophila melanogaster

We have analyzed at the molecular level diepoxybutane-induced mutants determined to have lesions affecting expression of the ry locus. Of the 21 mutants analyzed here, genetic analysis suggested that five were putative deficiencies involving ry and adjacent lethal loci. However, molecular analysis confirmed that only two of these five putative deficiencies were in fact deletions detectable by the methods used in the analysis. The remaining 16 mutants were viable as homozygotes, suggesting that their lesions were confined to the ry locus. Seven of these 16 intragenic mutants were determined to be deletions of genetic material as evidenced by altered restriction patterns relative to the wild type patterns. Thus, nine of 21 (43%) diepoxybutane-induced mutants are due to deletions ranging in size from approximately 50 base pairs to more than 8 kilobase pairs. Most of the deletions (seven of nine or 78%) are intragenic and less than 250 base pairs in size; it seems that most, if not all, affect coding rather than regulatory sequences.     

Genetic engineering of the multicellular green alga Volvox: a modified and multiplied bacterial antibiotic resistance gene as a dominant selectable marker

The green alga Volvox represents the simplest multicellular organism: Volvax is composed of only two cell types, somatic and reproductive. Volvox, therefore, is an attractive model system for studying various aspects of multicellularity. With the biolistic nuclear transformation of Volvox carteri, the powerful molecular genetic manipulation of this organism has been established, but applications have been restricted to an auxotrophic mutant serving as the DNA recipient. Therefore, a dominant selectable marker working in all strains and mutants of this organism is required. Among several gene constructs tested, the most advantageous results were obtained with a chimeric gene composed of the coding sequence of the bacterial ble gene, conferring resistance to the antibiotic zeocin, modified with insertions of two endogenous introns from the Volvox arylsulfatase gene and fused to 5' and 3' untranslated regions from the Volvox beta 2-tubulin gene. In the most suitable plasmid used, the gene dosage was increased 16-fold by a technique that allows exponential multiplication of a DNA fragment. Co-transformation of this plasmid and a non-selectable plasmid allowed the identification of zeocin resistant transformants with nuclear integration of both selectable and non-selectable plasmids. Stable expression of the ble gene and of genes from several non-selectable plasmids is demonstrated. The modified ble gene provides the first dominant marker for transformation of both wild-type and mutant strains of Volvox.     

Comprehensive genetic analysis of adhesin proteins and their role in virulence of Candida albicans

Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans’ ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, and every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this study yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.     

The synthetic genetic network around PKC1 identifies novel modulators and components of protein kinase C signaling in Saccharomyces cerevisiae

Budding yeast Saccharomyces cerevisiae contains one protein kinase C (PKC) isozyme encoded by the essential gene PKC1. Pkc1 is activated by the small GTPase Rho1 and plays a central role in the cell wall integrity (CWI) signaling pathway. This pathway acts primarily to remodel the cell surface throughout the normal life cycle and upon various environmental stresses. The pathway is heavily branched, with multiple nonessential branches feeding into and out of the central essential Rho1-Pkc1 module. In an attempt to identify novel components and modifiers of CWI signaling, we determined the synthetic lethal genetic network around PKC1 by using dominant-negative synthetic genetic array analysis. The resulting mutants are hypersensitive to lowered Pkc1 activity. The corresponding 21 nonessential genes are closely related to CWI function: 14 behave in a chemical-genetic epistasis test as acting in the pathway, and 6 of these genes encode known components. Twelve of the 21 null mutants display elevated CWI reporter activity, consistent with the idea that the pathway is activated by and compensates for loss of the gene products. Four of the 21 mutants display low CWI reporter activity, consistent with the idea that the pathway is compromised in these mutants. One of the latter group of mutants lacks Ack1(Ydl203c), an uncharacterized SEL-1 domain-containing protein that we find modulates pathway activity. Epistasis analysis places Ack1 upstream of Pkc1 in the CWI pathway and dependent on the upstream Rho1 GTP exchange factors Rom2 and Tus1. Overall, the synthetic genetic network around PKC1 directly and efficiently identifies known and novel components of PKC signaling in yeast.     

Morphogenetically Specific Mutability in DROSOPHILA ANANASSAE

A stock exhibiting hypermutability with respect to visible mutants (Om) affecting optic morphology was subjected to genetic analysis. The production of Om mutants, independently recovered with a frequency of two per 10⁴, is restricted to females and depends primarily on homozygosity of their X chromosomes; in heterozygotes, Om mutability is stimulated by the presence of either one of two extrachromosomally replicating elements previously identified in other stocks having cryptic mutability systems. The semidominant and nonpleiotropic Om mutants are not associated with gross rearrangements and they map to at least 15 loci. Most of the loci defined by mapping are represented by two or more Om mutants which, despite considerable interlocus mimicry, sometimes display locus-specific phenotypes. Om mutants are moderately unstable, and they are subject to dominant suppressors that arise spontaneously at either of two X-linked loci. An interpretation of these observations invokes an X-linked transposable element (tom) that specifically inserts into control sequences shared by a set of structural genes involved in eye morphogenesis.     

Instability at the k2 Mdh1-n y20 chromosomal region in soybean

Ten mutants have been reported at the k2 (tan saddle seed coat) Mdh1-n (mitochondrial malate dehydrogenase 1 null) y20 (yellow foliage) chromosomal region in soybean [Glycine max (L.) Merr.]. The precise genetic mechanism(s) responsible for generating these mutants is (are) not known. The objective of this study was to determine whether chromosomal instability exists at this region. We introduced the w4-m and Y18-m mutable systems into the three independent sources of tan saddle seed coat mutants, T239 (k2), T261 (k2 Mdh1-n), and L67-3483 (k2). A total of 12 bright yellow mutants were isolated with tan saddle seed coat, malate dehydrogenase 1 null phenotypes. Of these, 11 were found in 11 F2 mutant families out of a total of 977 derived by crossing T239 (k2), T261 (k2 Mdh1-n), and L67-3483 (k2) with six lines suspected to contain active transposable elements. One was found in the F3 generation derived from the cross A1937?×?T239 (k2). Of the 11 F2 mutant families, 10 (out of a total of 381 F2 families) were associated with the T239 (k2) genetic background, and one out of 323 was associated with the T261 (k2 Mdh1-n) genetic background. But no mutation events were found among the 273 families with the L67-3483 (k2) genetic background. Allelism and inheritance studies indicated that these 12 bright yellow mutants were new mutants in the k2 Mdh1-n y20 chromosomal region. Thus, on introducing the w4-m and Y18-m mutable systems into T239 (k2) and T261 (k2 Mdh1-n) genetic backgrounds, chromosomal instability was induced in this region. In addition, 21 greenish yellow mutants were identified in the total of 977 F2 families. All 21 greenish yellow mutants were associated with the T239 (k2) genetic background. The mutations for greenish yellow foliage affected foliage color only at the seedling stage. Cosegregation of the tan saddle seed coat character with greenish yellow foliage were observed for these 21 greenish yellow mutants, suggesting that the greenish yellow phenotype may be due to a pleiotropic effect of the k2 allele in T239 or to chromosomal rearrangements at or near the k2 allele in T239. Finally, we believe that the genetic mechanism responsible for this high frequency of instability at the k2 Mdh1-n y20 chromosomal region involves receptor element activities present at this chromosomal region, which may contain complex chromosomal rearrangements in T239 and T261.     

Wax constituents on the inflorescence stems of double eceriferum mutants in Arabidopsis reveal complex gene interactions

To shed new light on gene involvement in plant cuticular-wax production, 11 eceriferum (cer) mutants of Arabidopsis having dramatic alterations in wax composition of inflorescence stems were used to create 14 double cer mutants each with two homozygous recessive cer loci. A comprehensive analysis of stem waxes on these double mutants revealed unexpected CER gene interactions and new ideas about individual CER gene functions. Five of the 14 double cer mutants produced significantly more total wax than one of their respective cer parents, indicating from a genetic standpoint a partial bypassing (or complementation) of one cer mutation by the other. Eight of the 14 double cer mutants had alkane amounts lower than both respective cer parents, suggesting that most of these CER gene products play a major additive role in alkane synthesis. Other results suggested that some CER genes function in more than one step of the wax pathway, including those associated with sequential steps in acyl-CoA elongation. Surprisingly, complete epistasis was not observed for any of the cer gene combinations tested. Significant overlap or redundancy of genetic operations thus appears to be a central feature of wax metabolism. Future studies of CER gene product function, as well as the utilization of CER genes for crop improvement, must now account for the complex gene interactions described here.     

Regulation of the plastochron by three many-noded dwarf genes in barley

The plastochron, the time interval between the formation of two successive leaves, is an important determinant of plant architecture. We genetically and phenotypically investigated many-noded dwarf (mnd) mutants in barley. The mnd mutants exhibited a shortened plastochron and a decreased leaf blade length, and resembled previously reported plastochron1 (pla1), pla2, and pla3 mutants in rice. In addition, the maturation of mnd leaves was accelerated, similar to pla mutants in rice. Several barley mnd alleles were derived from three genes—MND1, MND4, and MND8. Although MND4 coincided with a cytochrome P450 family gene that is a homolog of rice PLA1, we clarified that MND1 and MND8 encode an N-acetyltransferase-like protein and a MATE transporter-family protein, which are respectively orthologs of rice GW6a and maize BIGE1 and unrelated to PLA2 or PLA3. Expression analyses of the three MND genes revealed that MND1 and MND4 were expressed in limited regions of the shoot apical meristem and leaf primordia, but MND8 did not exhibit a specific expression pattern around the shoot apex. In addition, the expression levels of the three genes were interdependent among the various mutant backgrounds. Genetic analyses using the double mutants mnd4mnd8 and mnd1mnd8 indicated that MND1 and MND4 regulate the plastochron independently of MND8, suggesting that the plastochron in barley is controlled by multiple genetic pathways involving MND1, MND4, and MND8. Correlation analysis between leaf number and leaf blade length indicated that both traits exhibited a strong negative association among different genetic backgrounds but not in the same genetic background. We propose that MND genes function in the regulation of the plastochron and leaf growth and revealed conserved and diverse aspects of plastochron regulation via comparative analysis of barley and rice.     

Physical and genetic organization of Petite and Grande yeast mitochondrial DNA. II. DNA-DNA hybridization studies and buoyant density determinations

Buoyant density of mitochondrial DNA from 14 cytoplasmic petite mutants issued from the same grande yeast Saccharomyces cerevisiae was determined. Mutants that have retained the mitochondrial gene conferring resistance to erythromycin displayed higher buoyant density, while mutants that have retained the mitochondrial gene conferring resistance to chloramphenicol displayed lower buoyant density. It is inferred that the segment which carries the E^R gene has a higher G + C content than the segment which carries the C^R gene. DNA-DNA filter hybridizations were carried out systematically in different reciprocal pair-wise combinations between mtDNAs purified from various mutants and from the grande. All petites were found to be deleted in 42 to 93% of the grande sequence, depending on the mutant studied. Sequence homology between petite mtDNAs was greatest in mutants retaining common genetic markers and was least when different genetic markers were retained. Practically no hybridization was found between some C^RE^O and C^OE^R mutants. Correlations established between the extent of DNA-DNA hybridization, kinetic and genetic complexity show that a selective enrichment of gene specific sequences occurs in mtDNA of petites.     

Genetics and function of DNA ligase in Escherichia coli

The characterization of two classes of DNA ligase mutants in Escherichia coli is described. The first class consists of three mutations coding for a temperature-sensitive ligase and defines the structural gene for DNA ligase (lig). The second class of mutants (lop) overproduces an apparently wild-type enzyme; a genetic diploid analysis implies that these are promoter or operator mutations, lig and lop are cotransduced by phage P1 and map at 46 minutes on the E. coli map. Detailed studies of two lig mutants (lig4 and lig ts7) are reported, lig ts7 is a conditionally lethal mutation, proving the essential nature of the ligase gene product. Neither mutant has a major defect in recombination or ultraviolet-repair, but both show retarded sealing of 10 S pulse-labeled DNA (Okazaki fragments).     

No influence of Indy on lifespan in Drosophila after correction for genetic and cytoplasmic background effects

To investigate whether alterations in mitochondrial metabolism affect longevity in Drosophila melanogaster, we studied lifespan in various single gene mutants, using inbred and outbred genetic backgrounds. As positive controls we included the two most intensively studied mutants of Indy, which encodes a Drosophila Krebs cycle intermediate transporter. It has been reported that flies heterozygous for these Indy mutations, which lie outside the coding region, show almost a doubling of lifespan. We report that only one of the two mutants lowers mRNA levels, implying that the lifespan extension observed is not attributable to the Indy mutations themselves. Moreover, neither Indy mutation extended lifespan in female flies in any genetic background tested. In the original genetic background, only the Indy mutation associated with altered RNA expression extended lifespan in male flies. However, this effect was abolished by backcrossing into standard outbred genetic backgrounds, and was associated with an unidentified locus on the X chromosome. The original Indy line with long-lived males is infected by the cytoplasmic symbiont Wolbachia, and the longevity of Indy males disappeared after tetracycline clearance of this endosymbiont. These findings underscore the critical importance of standardisation of genetic background and of cytoplasm in genetic studies of lifespan, and show that the lifespan extension previously claimed for Indy mutants was entirely attributable to confounding variation from these two sources. In addition, we saw no effects on lifespan of expression knockdown of the Indy orthologues nac-2 and nac-3 in the nematode Caenorhabditis elegans.     

Insights on ADAMTS proteases and ADAMTS-like proteins from mammalian genetics

The mammalian ADAMTS superfamily comprises 19 secreted metalloproteinases and 7 ADAMTS-like proteins, each the product of a distinct gene. Thus far, all appear to be relevant to extracellular matrix function or to cell–matrix interactions. Most ADAMTS functions first emerged from analysis of spontaneous human and animal mutations and genetically engineered animals. The clinical manifestations of Mendelian disorders resulting from mutations in ADAMTS2, ADAMTS10, ADAMTS13, ADAMTS17, ADAMTSL2 and ADAMTSL4 identified essential roles for each gene, but also suggested potential cooperative functions of ADAMTS proteins. These observations were extended by analysis of spontaneous animal mutations, such as in bovine ADAMTS2, canine ADAMTS10, ADAMTS17 and ADAMTSL2 and mouse ADAMTS20. These human and animal disorders are recessive and their manifestations appear to result from a loss-of-function mechanism. Genome-wide analyses have determined an association of some ADAMTS loci such as ADAMTS9 and ADAMTS7, with specific traits and acquired disorders. Analysis of genetically engineered rodent mutations, now achieved for over half the superfamily, has provided novel biological insights and animal models for the respective human genetic disorders and suggested potential candidate genes for related human phenotypes. Engineered mouse mutants have been interbred to generate combinatorial mutants, uncovering cooperative functions of ADAMTS proteins in morphogenesis. Specific genetic models have provided crucial insights on mechanisms of osteoarthritis (OA), a common adult-onset degenerative condition. Engineered mutants will facilitate interpretation of exome variants identified in isolated birth defects and rare genetic conditions, as well as in genome-wide screens for trait and disease associations. Mammalian forward and reverse genetics, together with genome-wide analysis, together constitute a powerful force for revealing the functions of ADAMTS proteins in physiological pathways and health disorders. Their continuing use, together with genome-editing technology and the ability to generate stem cells from mutants, presents numerous opportunities for advancing basic knowledge, human disease pathways and therapy.     

Nitrate-nonutilizing mutants ofGibberella zeae (Fusarium graminearum) and their use in determining vegetative compatibility

Twenty-four single-spore isolates ofFusarium graminearum were obtained from scabby wheat seeds or glumes collected from 23 locations in Kansas in 1990. All isolates were sexually fertile and homothallic. Nitrate-nonutilizing (nit) mutants of each isolate were generated on a medium amended with 1.5% KCIO₃. Of 378 mutants, 161 were able to utilize nitrite and hypoxanthine (nit1), 165 utilized hypoxanthine but not nitrite (nit3), 47 utilized nitrite but not hypoxanthine (NitM), and 5 appeared to be global nitrogen regulatory mutants similar to the previously describednnu mutant. Complementation was tested by pairingnit1 mutants of each isolate with either a NitM or anit3 mutant from each isolate on media containing nitrate as the sole nitrogen source. Complementation was more pronounced whennit1 and NitM mutants were paired. Mutants were only able to complement with other mutants from the same wild-type isolate. Therefore, each wild-type isolate belonged to a genetically distinct vegetative compatibility group. The genetic diversity suggests that sexual genetic recombination may be important in the field.     

Probable exclusion of the cortexin-encoding gene as a candidate for mouse neurological mutants: nervous,tottering and motor neuron degeneration

We have tested the gene encoding cortexin, Ctxn, which maps to chromosome 8, as a candidate for the mouse neurological mutants: nervous (nr), tottering (tg) plus tottering-leaner (tg^la), and motor neuron degeneration (mnd) by Northern blot analysis of brain poly(A)⁺ RNA and direct polymerase chain reaction (PCR) sequencing. No difference from wild-type was seen in any of these mutants. Based upon these observations, we conclude that Ctxn is not involved in the genetic defects found in nr, tg or mnd mice.