Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. donovani (TUBA5) that is deficient in LdNT1 transport and consequently resistant to tubercidin. In TUBA5 cells, the LdNT1.2 genes had the same sequence as wild-type cells. However, because LdNT1.2 mRNA is not detectable in either wild-type or TUBA5 promastigotes, LdNT1.2 does not contribute to nucleoside transport in this stage of the life cycle. In contrast, the TUBA5 cells were compound heterozygotes at the LdNT1.1 locus containing two mutant alleles that encompassed distinct point mutations, each of which impaired transport function. One of the mutant LdNT1.1 alleles encoded a G183D substitution in predicted TM 5, and the other allele contained a C337Y change in predicted TM 7. Whereas G183D and C337Y mutants had only slightly elevated adenosine Km values, the severe impairment in transport resulted from drastically (≈20-fold) reduced Vmax values. Because these transporters were correctly targeted to the plasma membrane, the reduction in Vmax apparently resulted from a defect in translocation. Strikingly, G183 was essential for pyrimidine nucleoside but not adenosine transport. A mutant transporter with a G183A substitution had an altered substrate specificity, exhibiting robust adenosine transport but undetectable uridine uptake. These results suggest that TM 5 is likely to form part of the nucleoside translocation pathway in LdNT1.1

A Tropomyosin-2 Mutation Suppresses a Troponin I Myopathy in Drosophila

A suppressor mutation, D53, of the held-up2 allele of the Drosophila melanogaster Troponin I (wupA) gene is described. D53, a missense mutation, S185F, of the tropomyosin-2, Tm2, gene fully suppresses all the phenotypic effects of held-up2, including the destructive hypercontraction of the indirect flight muscles (IFMs), a lack of jumping, the progressive myopathy of the walking muscles, and reductions in larval crawling and feeding behavior. The suppressor restores normal function of the IFMs, but flight ability decreases with age and correlates with an unusual, progressive structural collapse of the myofibrillar lattice starting at the center. The S185F substitution in Tm2 is close to a troponin T binding site on tropomyosin. Models to explain suppression by D53, derived from current knowledge of the vertebrate troponin-tropomyosin complex structure and functions, are discussed. The effects of S185F are compared with those of two mutations in residues 175 and 180 of human α-tropomyosin 1 which cause familial hypertrophic cardiomyopathy (HCM).

Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays

We describe a method to screen pools of DNA from multiple transposon lines for insertions in many genes simultaneously. We use thermal asymmetric interlaced–PCR, a hemispecific PCR amplification protocol that combines nested, insertion-specific primers with degenerate primers, to amplify DNA flanking the transposons. In reconstruction experiments with previously characterized Arabidopsis lines carrying insertions of the maize Dissociation (Ds) transposon, we show that fluorescently labeled, transposon-flanking fragments overlapping ORFs hybridize to cognate expressed sequence tags (ESTs) on a DNA microarray. We further show that insertions can be detected in DNA pools from as many as 100 plants representing different transposon lines and that all of the tested, transposon-disrupted genes whose flanking fragments can be amplified individually also can be detected when amplified from the pool. The ability of a transposon-flanking fragment to hybridize declines rapidly with decreasing homology to the spotted DNA fragment, so that only ESTs with >90% homology to the transposon-disrupted gene exhibit significant cross-hybridization. Because thermal asymmetric interlaced–PCR fragments tend to be short, use of the present method favors recovery of insertions in and near genes. We apply the technique to screening pools of new Ds lines using cDNA microarrays containing ESTs for ≈1,000 stress-induced and -repressed Arabidopsis genes.

Ullrich scleroatonic muscular dystrophy is caused by recessive mutations in collagen type VI

Ullrich syndrome is a recessive congenital muscular dystrophy affecting connective tissue and muscle. The molecular basis is unknown. Reverse transcription–PCR amplification performed on RNA extracted from fibroblasts or muscle of three Ullrich patients followed by heteroduplex analysis displayed heteroduplexes in one of the three genes coding for collagen type VI (COL6). In patient A, we detected a homozygous insertion of a C leading to a premature termination codon in the triple-helical domain of COL6A2 mRNA. Both healthy consanguineous parents were carriers. In patient B, we found a deletion of 28 nucleotides because of an A → G substitution at nucleotide −2 of intron 17 causing the activation of a cryptic acceptor site inside exon 18. The second mutation was an exon skipping because of a G → A substitution at nucleotide −1 of intron 23. Both mutations are present in an affected brother. The first mutation is also present in the healthy mother, whereas the second mutation is carried by their healthy father. In patient C, we found only one mutation so far—the same deletion of 28 nucleotides found in patient B. In this case, it was a de novo mutation, as it is absent in her parents. mRNA and protein analysis of patient B showed very low amounts of COL6A2 mRNA and of COL6. A near total absence of COL6 was demonstrated by immunofluorescence in fibroblasts and muscle. Our results demonstrate that Ullrich syndrome is caused by recessive mutations leading to a severe reduction of COL6.

Systematic Reverse Genetics of Transfer-DNA-Tagged Lines of Arabidopsis1 : Isolation of Mutations in the Cytochrome P450 Gene Superfamily

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

Nuclear-Gene Mutations Suppress a Defect in the Expression of the Chloroplast-Encoded Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase1

The green alga Chlamydomonas reinhardtii mutant 76–5EN lacks photosynthesis because of a nuclear-gene mutation that specifically inhibits expression of the chloroplast gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39). Photosynthesis-competent revertants were selected from mutant 76–5EN to explore the possibility of increasing Rubisco expression. Genetic analysis of 10 revertants revealed that most arose from suppressor mutations in nuclear genes distinct from the original 76–5EN mutant gene. The revertant strains have regained various levels of Rubisco holoenzyme, but none of the suppressor mutations increased Rubisco expression above the wild-type level in either the presence or absence of the 76–5EN mutation. One suppressor mutation, S107–4B, caused a temperature-conditional, photosynthesis-deficient phenotype in the absence of the original 76–5EN mutation. The S107–4B strain was unable to grow photosynthetically at 35°C, but it expressed a substantial level of Rubisco holoenzyme. Whereas the 76–5EN gene encodes a nuclear factor that appears to be required for the transcription of the Rubisco large-subunit gene, the S107–4B nuclear gene may be required for the expression of other chloroplast genes.

Transit Peptide Mutations That Impair in Vitro and in Vivo Chloroplast Protein Import Do Not Affect Accumulation of the γ-Subunit of Chloroplast ATPase1

We have begun to take a genetic approach to study chloroplast protein import in Chlamydomonas reinhardtii by creating deletions in the transit peptide of the γ-subunit of chloroplast ATPase-coupling factor 1 (CF1-γ, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into an atpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF1-γ accumulation in vivo. The CF1-γ transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF1-γ.

Effects of mutations involving cell division, recombination, and chromosome dimer resolution on a priA2∷kan mutant

Recombinational repair of replication forks can occur either to a crossover (XO) or noncrossover (non-XO) depending on Holliday junction resolution. Once the fork is repaired by recombination, PriA is important for restarting these forks in Escherichia coli. PriA mutants are Rec− and UV sensitive and have poor viability and 10-fold elevated basal levels of SOS expression. PriA sulB mutant cells and their nucleoids were studied by differential interference contrast and fluorescence microscopy of 4′,6-diamidino-2-phenylindole-stained log phase cells. Two populations of cells were seen. Eighty four percent appeared like wild type, and 16% of the cells were filamented and had poorly partitioned chromosomes (Par−). To probe potential mechanisms leading to the two populations of cells, mutations were added to the priA sulB mutant. Mutating sulA or introducing lexA3 decreased, but did not eliminate filamentation or defects in partitioning. Mutating either recA or recB virtually eliminated the Par− phenotype. Filamentation in the recB mutant decreased to 3%, but increased to 28% in the recA mutant. The ability to resolve and/or branch migrate Holliday junctions also appeared crucial in the priA mutant because removing either recG or ruvC was lethal. Lastly, it was tested whether the ability to resolve chromosome dimers caused by XOs was important in a priA mutant by mutating dif and the C-terminal portion of ftsK. Mutation of dif showed no change in phenotype whereas ftsK1∷cat was lethal with priA2∷kan. A model is proposed where the PriA-independent pathway of replication restart functions at forks that have been repaired to non-XOs.