Molecular relationships between alcohol dehydrogenase null-activity alleles from natural populations of Drosophila melanogaster.

Alcohol dehydrogenase null-activity alleles extracted from a number of natural populations of Drosophila melanogaster in Tasmania were shown to be molecularly similar by probing, with an oligonucleotide specific to an inserted region in intron 2 of the gene, genomic DNA amplified by the polymerase chain reaction. This insertion had previously been shown to be the cause of the loss of activity in one of the null alleles whose DNA sequence was known. Three Adh null alleles from mainland populations did not contain the insertion. Two of these null alleles, extracted from the Coffs Harbour population in different years, were cloned, and their DNA sequences showed that they were identical and that both had a 438-bp deletion which removed most of exon 2. The third null allele, identified in a sample of flies from Chateau Tahbilk, was shown by 4-bp restriction-endonuclease mapping to contain a 320-bp insertion in intron 1, although this may not be the cause of the loss of activity. The data show that at least three different Adh null alleles have been found in Australian populations and that at least two have been maintained as heterozygotes over a period of years.     

Null alleles at two lactate dehydrogenase loci in rainbow trout are associated with decreased developmental stability

Previous studies with rainbow trout (Oncorhynchus mykiss) have shown that allozymic heterozygotes have increased developmental stability, as measured by reduced fluctuating bilateral asymmetry. In this paper, we examine the phenotypic effects of null alleles at two lactate dehydrogenase (LDH) loci. If the association between allozymic heterozygosity and developmental stability is due largely to linked chromosomal segments, then we would expect null allele heterozygotes to have increased developmental stability. In contrast, heterozygotes for LDH null alleles in three populations have reduced developmental stability. This suggests that the reduction in enzyme activity at these loci is having a deleterious effect on development that is strong enough to mask any beneficial effects that may be associated with heterozygosity for these chromosomal segments. The LDH loci examined in this study are members of two different paralogous pairs of duplicate genes produced by the polyploidization of the ancestral salmonid genome. The apparent deleterious effects of these null alleles in heterozygotes could retard the possible loss of duplicate gene expression.     

Effects of microsatellite null alleles on assignment testing

Microsatellite null alleles are found to a varying degree across all taxa. They are problematic as they may inflate measures of genetic differentiation and create false homozygotes. Although there are several methods for correcting allele frequencies for null alleles and enable estimations of F(ST), much less is known about how null alleles affect assignment testing. Data presented here, based on simulations, show that the percentage of correctly assigned individuals in model-based clustering and Bayesian assignment methods were slightly, though significantly, reduced in the presence of null alleles (frequency range from 0.000 to 0.913). The bias in assignment tests caused by null alleles lead to a slight reduction in the power to correctly assigned individuals (0.2 and 1.0 percent units for STRUCTURE- and 2.4 percent units for GENECLASS-based assignment tests). Further, the presence of null alleles caused a small, however, significant overestimation of F(ST). Consequently, microsatellite loci affected by null alleles would probably not alter the overall outcome of assignment testing and could therefore be included in these types of studies. Nevertheless, loci prone to null alleles should be used with caution as they lower the power of assignment tests and alter the accuracy of F(ST), and loci less prone to null alleles should always be preferred.     

Biochemical and molecular analysis of spontaneous and induced mutations at the mouse Mod-1 locus

We have analyzed five Mod-1 (malic enzyme) mutants at the molecular and biochemical level. Four of these mutants, three electrophoretic variants and one null mutant, were induced by ethylnitrosourea (ENU). Another null mutant was the result of a spontaneous mutation. All of these mutations were heritable in a Mendelian fashion and viable in the homozygous condition. Restriction endonuclease and Southern blot analysis revealed that the spontaneous null mutant possessed an altered restriction fragment banding pattern. All of the ENU-induced mutants possessed normal restriction fragment banding patterns. All 5 mutants produced normal levels of Mod-1-specific mRNA. Only the spontaneous null mutant produced mRNA with altered size, which was consistent with the altered DNA-banding pattern. MOD-1 enzyme activity levels were normal in the three ENU-induced mutants with altered electrophoretic mobility. Enzyme activity was significantly lower than normal in tissues from animals homozygous for the null alleles, however, using Western blot analysis, low but significant levels of MOD-1 protein in Mod-1 null homozygotes were detected.     

Effects of XeCl UV308 nm laser radiation on survival and mutability of recA-proficient and recA-defective Escherichia coli strains

recA1, recA13 and recA56 are considered null alleles of the Escherichia coli recA gene because they were shown to have essentially no activity in vivo. In this study, we used strains harboring the recA null alleles and their recA-proficient congenic counterpart to assess the lethal and the mutagenic effects elicited by near-UV(308 nm) coherent radiation generated by a XeCl excimer laser. We compared these effects with those produced by a conventional far-UV(254 nm) germicidal lamp. Compared to the germicidal lamp, the excimer laser was able to better discriminate the different recA-defective strains on the basis of their UV-radiation sensitivity, which was progressively higher in the strains with the alleles in the order recA1, recA56 and recA13. This finding was consistent with previous data on residual biochemical activities of the respective mutated RecA proteins in vitro. The discrepancy between the results obtained with the lamp and laser irradiation suggested that the biological response to the two radiations involves distinct mechanisms. This hypothesis was supported by the evidence that exposure to near-UV(308 nm) radiation induced mutagenesis in recA-defective strains at an extent considerably greater than in recA-proficient strains. In contrast, far-UV(254 nm)-radiation-induced mutagenesis was reported to be largely dependent on a functional recA allele.     

In vivo characterization of Escherichia coli ftsZ mutants: effects on Z-ring structure and function

We have characterized the in vivo phenotypes of 17 mutations of Escherichia coli ftsZ. In particular, we determined whether these mutations can complement a null ftsZ phenotype, and we demonstrated that two noncomplementing mutations show partial dominant-negative behavior. We performed immunofluorescence microscopy to determine whether these mutants could assemble into normal or abnormal structures in vivo. The mutants separated into four classes-those that complemented the null and formed normal FtsZ rings, those that complemented the null but formed aberrant FtsZ structures, those that formed aberrant FtsZ structures and did not complement, and those that were unable to form any FtsZ structures. We did not find any mutations that produced nonfunctional Z rings of normal appearance. Surprisingly, some mutants that produced extensively spiraled Z-ring structures divided and grew with a normal doubling time. The analysis was carried out using a complementation system based on an ftsZ deletion strain, a temperature-sensitive rescue plasmid, and a complementation vector that placed mutated ftsZ alleles under the control of the pBAD promoter, which offered several advantages over previous systems.     

‘True’ null allele detection in microsatellite loci: a comparison of methods,assessment of difficulties and survey of possible improvements

Null alleles are alleles that for various reasons fail to amplify in a PCR assay. The presence of null alleles in microsatellite data is known to bias the genetic parameter estimates. Thus, efficient detection of null alleles is crucial, but the methods available for indirect null allele detection return inconsistent results. Here, our aim was to compare different methods for null allele detection, to explain their respective performance and to provide improvements. We applied several approaches to identify the ‘true’ null alleles based on the predictions made by five different methods, used either individually or in combination. First, we introduced simulated ‘true’ null alleles into 240 population data sets and applied the methods to measure their success in detecting the simulated null alleles. The single best‐performing method was ML‐NullFreq_frequency. Furthermore, we applied different noise reduction approaches to improve the results. For instance, by combining the results of several methods, we obtained more reliable results than using a single one. Rule‐based classification was applied to identify population properties linked to the false discovery rate. Rules obtained from the classifier described which population genetic estimates and loci characteristics were linked to the success of each method. We have shown that by simulating ‘true’ null alleles into a population data set, we may define a null allele frequency threshold, related to a desired true or false discovery rate. Moreover, using such simulated data sets, the expected null allele homozygote frequency may be estimated independently of the equilibrium state of the population.     

Deficient and Null Variants of SERPINA1 Are Proteotoxic in a Caenorhabditis elegans Model of α1-Antitrypsin Deficiency

α1-antitrypsin deficiency (ATD) predisposes patients to both loss-of-function (emphysema) and gain-of-function (liver cirrhosis) phenotypes depending on the type of mutation. Although the Z mutation (ATZ) is the most prevalent cause of ATD, >120 mutant alleles have been identified. In general, these mutations are classified as deficient (<20% normal plasma levels) or null (<1% normal levels) alleles. The deficient alleles, like ATZ, misfold in the ER where they accumulate as toxic monomers, oligomers and aggregates. Thus, deficient alleles may predispose to both gain- and loss-of-function phenotypes. Null variants, if translated, typically yield truncated proteins that are efficiently degraded after being transiently retained in the ER. Clinically, null alleles are only associated with the loss-of-function phenotype. We recently developed a C. elegans model of ATD in order to further elucidate the mechanisms of proteotoxicity (gain-of-function phenotype) induced by the aggregation-prone deficient allele, ATZ. The goal of this study was to use this C. elegans model to determine whether different types of deficient and null alleles, which differentially affect polymerization and secretion rates, correlated to any extent with proteotoxicity. Animals expressing the deficient alleles, Mmalton, Siiyama and S (ATS), showed overall toxicity comparable to that observed in patients. Interestingly, Siiyama expressing animals had smaller intracellular inclusions than ATZ yet appeared to have a greater negative effect on animal fitness. Surprisingly, the null mutants, although efficiently degraded, showed a relatively mild gain-of-function proteotoxic phenotype. However, since null variant proteins are degraded differently and do not appear to accumulate, their mechanism of proteotoxicity is likely to be different to that of polymerizing, deficient mutants. Taken together, these studies showed that C. elegans is an inexpensive tool to assess the proteotoxicity of different AT variants using a transgenic approach.     

Disruption of C-terminal cytoplasmic domain of betaPS integrin subunit has dominant negative properties in developing Drosophila

We have analyzed a set of new and existing strong mutations in the myospheroid gene, which encodes the betaPS integrin subunit of Drosophila. In addition to missense and other null mutations, three mutants behave as antimorphic alleles, indicative of dominant negative properties. Unlike null alleles, the three antimorphic mutants are synthetically lethal in double heterozygotes with an inflated (alphaPS2) null allele, and they fail to complement very weak, otherwise viable alleles of myospheroid. Two of the antimorphs result from identical splice site lesions, which create a frameshift in the C-terminal half of the cytoplasmic domain of betaPS. The third antimorphic mutation is caused by a stop codon just before the cytoplasmic splice site. These mutant betaPS proteins can support cell spreading in culture, especially under conditions that appear to promote integrin activation. Analyses of developing animals indicate that the dominant negative properties are not a result of inefficient surface expression, or simple competition between functional and nonfunctional proteins. These data indicate that mutations disrupting the C-terminal cytoplasmic domain of integrin beta subunits can have dominant negative effects in situ, at normal levels of expression, and that this property does not necessarily depend on a specific new protein sequence or structure. The results are discussed with respect to similar vertebrate beta subunit cytoplasmic mutations.