Germ line deletion of the CD1 locus exacerbates diabetes in the NOD mouse

Quantitative and qualitative defects in CD1-restricted natural killer T cells have been reported in several autoimmune-prone strains of mice, including the nonobese diabetic (NOD) mouse. These defects are believed to be associated with the emergence of spontaneous autoimmunity. Here we demonstrate that both CD1d-null NOD and CD1d-null NOD/BDC2.5 T cell receptor transgenic mice have an accelerated onset and increased incidence of diabetes when compared with CD1d+/− and CD1d+/+ littermates. The acceleration of disease did not seem to result from changes in the T helper (Th)1/Th2 balance because lymphocytes purified from lymphoid organs and pancreatic islets of wild-type and CD1d-null mice secreted equivalent amounts of IFN-γ and IL-4 after stimulation. In contrast, the pancreata of CD1d-null mice harbored significantly higher numbers of activated memory T cells expressing the chemokine receptor CCR4. Notably, the presence of these T cells was associated with immunohistochemical evidence of increased destructive insulitis. Thus, CD1d-restricted T cells are critically important for regulation of the spontaneous disease process in NOD mice.

Yeast Rab GTPase-activating Protein Gyp1p Localizes to the Golgi Apparatus and Is a Negative Regulator of Ypt1p

A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1Δ strain displays a growth defect on synthetic medium at 37°C. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1Δ cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1Δ cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.

Isolation and Characterization of Effector-Loop Mutants of CDC42 in Yeast

The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of “effector-loop” mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants of Saccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors.

New Arabidopsis cue Mutants Suggest a Close Connection between Plastid- and Phytochrome Regulation of Nuclear Gene Expression1

We searched for new components that are involved in the positive regulation of nuclear gene expression by light by extending a screen for Arabidopsis cue (chlorophyll a/b-binding [CAB] protein-underexpressed) mutants (H.-M. Li, K. Culligan, R.A. Dixon, J. Chory [1995] Plant Cell 7: 1599–1610). cue mutants display reduced expression of the CAB3 gene, which encodes light-harvesting chlorophyll protein, the main chloroplast antenna. The new mutants can be divided into (a) phytochrome-deficient mutants (hy1 and phyB), (b) virescent or delayed-greening mutants (cue3, cue6, and cue8), and (c) uniformly pale mutants (cue4 and cue9). For each of the mutants, the reduction in CAB expression correlates with the visible phenotype, defective chloroplast development, and reduced abundance of the light-harvesting chlorophyll protein. Levels of protochlorophyllide oxidoreductase (POR) were reduced to varying degrees in etiolated mutant seedlings. In the dark, whereas the virescent mutants displayed reduced CAB expression and the lowest levels of POR protein, the other mutants expressed CAB and accumulated POR at near wild-type levels. All of the mutants, with the exception of cue6, were compromised in their ability to derepress CAB expression in response to phytochrome activation. Based on these results, we propose that the previously postulated plastid-derived signal is closely involved in the pathway through which phytochrome regulates the expression of nuclear genes encoding plastid proteins.

Conditional Synergism between Cryptochrome 1 and Phytochrome B Is Shown by the Analysis of phyA, phyB, and hy4 Simple, Double, and Triple Mutants in Arabidopsis

Wild-type or phyA, phyB, or hy4 mutant Arabidopsis seedlings lacking phytochrome A (phyA), phytochrome B (phyB), or cryptochrome 1 (cry1), respectively, and the double and triple mutants were used in combination with blue-light treatments given simultaneously with red or far-red light. We investigated the interaction between phytochromes and cry1 in the control of hypocotyl growth and cotyledon unfolding. Under conditions deficient for cry1 (short exposures to blue light) or phyB (far-red background), these photoreceptors acted synergistically: Under short exposures to blue light (3 h/d) added to a red-light background, cry1 activity required phyB (e.g. the hy4 mutant was taller than the wild type but the phyBhy4 mutant was not taller than the phyB mutant). Under prolonged exposures to blue light (24 h/d) added to a far-red light background, phyB activity required cry1 (e.g. the phyAphyB mutant was taller than the phyA mutant but the phyAphyBhy4 mutant was not taller than the phyAhy4 mutant). Under more favorable light inputs, i.e. prolonged exposures to blue light added to a red-light background, the effects of cry1 and phyB were independent. Thus, the synergism between phyB and cry1 is conditional. The effect of cry1 was not reduced by the phyA mutation under any tested light condition. Under continuous blue light the triple mutant phyAphyBhy4 showed reduced hypocotyl growth inhibition and cotyledon unfolding compared with the phyAphyB mutant. The action of cry1 in the phyAphyB double mutant was higher under the red-light than the far-red-light background, indicating a synergistic interaction between cry1 and phytochromes C, D, or E; however, a residual action of cry1 independent of any phytochrome is likely to occur.

A New Class of Arabidopsis Mutants with Reduced Hexadecatrienoic Acid Fatty Acid Levels1

Chloroplast glycerolipids in a number of higher-plant species, including Arabidopsis thaliana, are synthesized by two distinct pathways termed the prokaryotic and eukaryotic pathways. The molecules of galactolipids produced by the prokaryotic pathway contain substantial amounts of hexadecatrienoic acid fatty acid. Here we describe a new class of mutants, designated gly1, with reduced levels of hexadecatrienoic acid. Lipid fatty acid profiles indicated that gly1 mutants exhibited a reduced carbon flux through the prokaryotic pathway that was compensated for by an increased carbon flux through the eukaryotic pathway. Genetic and biochemical approaches revealed that the gly1 phenotype could not be explained by a deficiency in the enzymes of the prokaryotic pathway. The flux of fatty acids into the prokaryotic pathway is sensitive to changes in glycerol-3-phosphate (G3P) availability, and the chloroplast G3P pool can be increased by exogenous application of glycerol to leaves. Exogenous glycerol treatment of gly1 plants allowed chemical complementation of the mutant phenotype. These results are consistent with a mutant lesion affecting the G3P supply within the chloroplast. The gly1 mutants may therefore help in determining the pathway for synthesis of chloroplast G3P.

Aluminum-Resistant Arabidopsis Mutants That Exhibit Altered Patterns of Aluminum Accumulation and Organic Acid Release from Roots1

Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L.V. Kochian [1998] Plant Physiol 117: 19–27).

An Embryo-Defective Mutant of Arabidopsis Disrupted in the Final Step of Biotin Synthesis

Auxotrophic mutants have played an important role in the genetic dissection of biosynthetic pathways in microorganisms. Equivalent mutants have been more difficult to identify in plants. The bio1 auxotroph of Arabidopsis thaliana was shown previously to be defective in the synthesis of the biotin precursor 7,8-diaminopelargonic acid. A second biotin auxotroph of A. thaliana has now been identified. Arrested embryos from this bio2 mutant are defective in the final step of biotin synthesis, the conversion of dethiobiotin to biotin. This enzymatic reaction, catalyzed by the bioB product (biotin synthase) in Escherichia coli, has been studied extensively in plants and bacteria because it involves the unusual addition of sulfur to form a thiophene ring. Three lines of evidence indicate that bio2 is defective in biotin synthase production: mutant embryos are rescued by biotin but not dethiobiotin, the mutant allele maps to the same chromosomal location as the cloned biotin synthase gene, and gel-blot hybridizations and polymerase chain reaction amplifications revealed that homozygous mutant plants contain a deletion spanning the entire BIO2-coding region. Here we describe how the isolation and characterization of this null allele have provided valuable insights into biotin synthesis, auxotrophy, and gene redundancy in plants.

An AU-box motif upstream of the SD sequence of light-dependent psbA transcripts confers mRNA instability in darkness in cyanobacteria

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. The expression of the psbA2 transcript has been shown to be light-dependent as assessed under light and dark (12/12 h) cycling conditions. We aligned the 5′-untranslated leader regions (UTRs) of psbAs from different photosynthetic organisms and identified a conserved sequence, UAAAUAAA or the ‘AU-box’, just upstream of the SD sequences. To clarify the role of 5′-upstream cis-elements containing the AU-box for light-dependent expression of psbA2, a series of deletion and point mutations in the region were introduced into the genome of heterologous cyanobacterium Synechococcus sp. strain PCC 7942, and psbA2 expression was examined. A clear pattern of light-dependent expression was observed in recombinant cyanobacteria carrying the K-81 psbA2 –38/+36 region (which includes the minimal promoter element and a light-dependent cis-element with the AU-box), +1 indicating the transcription start site. A constitutive pattern of expression, in which the transcripts remained almost stable under dark conditions, was obtained in cells harboring the –38/+14 region (the minimal element), indicating that the +14/+36 region with the AU-box is important for the observed light-dependent expression. Point mutations analyses within the AU-box also revealed that changes in number, direction and identity (as assayed by adenine/uridine nucleotide substitutions) influenced the light-dependent pattern of expression. The level of psbA2 transcripts increased markedly in CG- or deletion-box mutants in the dark, strongly indicating that the AU- (AT-) box acts as a negative cis-element. Furthermore, characterization of transcript accumulation in cells treated with rifampicin suggests that psbA2 5′-mRNA is unstable in the dark, supporting the view that the light-dependent expression is controlled at the post-transcriptional level. We discuss various mechanisms that may lead to altered mRNA stability such as the binding of factor(s) or ribosomes to the 5′-UTR and possible roles of the AU-box motif and the SD sequence.

Cloning the human and mouse MMS19 genes and functional complementation of a yeast mms19 deletion mutant

The MMS19 gene of the yeast Saccharomyces cerevisiae encodes a polypeptide of unknown function which is required for both nucleotide excision repair (NER) and RNA polymerase II (RNAP II) transcription. Here we report the molecular cloning of human and mouse orthologs of the yeast MMS19 gene. Both human and Drosophila MMS19 cDNAs correct thermosensitive growth and sensitivity to killing by UV radiation in a yeast mutant deleted for the MMS19 gene, indicating functional conservation between the yeast and mammalian gene products. Alignment of the translated sequences of MMS19 from multiple eukaryotes, including mouse and human, revealed the presence of several conserved regions, including a HEAT repeat domain near the C-terminus. The presence of HEAT repeats, coupled with functional complementation of yeast mutant phenotypes by the orthologous protein from higher eukaryotes, suggests a role of Mms19 protein in the assembly of a multiprotein complex(es) required for NER and RNAP II transcription. Both the mouse and human genes are ubiquitously expressed as multiple transcripts, some of which appear to derive from alternative splicing. The ratio of different transcripts varies in several different tissue types.

Subnuclear targeting of Runx/Cbfa/AML factors is essential for tissue-specific differentiation during embryonic development

Runx (Cbfa/AML) transcription factors are critical for tissue-specific gene expression. A unique targeting signal in the C terminus directs Runx factors to discrete foci within the nucleus. Using Runx2/CBFA1/AML3 and its essential role in osteogenesis as a model, we investigated the fundamental importance of fidelity of subnuclear localization for tissue differentiating activity by deleting the intranuclear targeting signal via homologous recombination. Mice homozygous for the deletion (Runx2ΔC) do not form bone due to maturational arrest of osteoblasts. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are indistinguishable from those of the homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated Runx2ΔC protein enters the nucleus and retains normal DNA binding activity, but shows complete loss of intranuclear targeting. These results demonstrate that the multifunctional N-terminal region of the Runx2 protein is not sufficient for biological activity. We conclude that subnuclear localization of Runx factors in specific foci together with associated regulatory functions is essential for control of Runx-dependent genes involved in tissue differentiation during embryonic development.

Enhanced antitumor efficacy of a herpes simplex virus mutant isolated by genetic selection in cancer cells

Replication-competent, attenuated herpes simplex virus-1 (HSV-1) derivatives that contain engineered mutations into the viral γ34.5 virulence gene have been used as oncolytic agents. However, as attenuated mutants often grow poorly, they may not completely destroy some tumors and surviving cancer cells simply regrow. Thus, although HSV-1 γ34.5 mutants can reduce the growth of human tumor xenografts in mice and have passed phase I safety studies, their efficacy is limited because they replicate poorly in many human tumor cells. Previously, we selected for a γ34.5 deletion mutant variant that regained the ability to replicate efficiently in tumor cells. Although this virus contains an extragenic suppressor mutation that confers enhanced growth in tumor cells, it remains attenuated. Here, we demonstrate that the suppressor virus replicates to greater levels in prostate carcinoma cells and, importantly, is a more potent inhibitor of tumor growth in an animal model of human prostate cancer than the γ34.5 parent virus. Thus, genetic selection in cancer cells can be used as a tool to enhance the antitumor activity of a replication-competent virus. The increased therapeutic potency of this oncolytic virus may be useful in the treatment of a wide variety of cancers.

A genetic selection for Caenorhabditis elegans synaptic transmission mutants.

We have isolated 165 Caenorhabditis elegans mutants, representing 21 genes, that are resistant to inhibitors of cholinesterase (Ric mutants). Since mutations in 20 of the genes appear not to affect acetylcholine reception, we suggest that reduced acetylcholine release contributes to the Ric phenotype of most Ric mutants. Mutations in 15 of the genes lead to defects in a gamma-aminobutyric acid-dependent behavior; these genes are likely to encode proteins with general, rather than cholinergic-specific, roles in synaptic transmission. Ten of the genes have been cloned. Seven encode homologs of proteins that function in the synaptic vesicle cycle: two encode cholinergic-specific proteins, while five encode general presynaptic proteins. Two other Ric genes encode homologs of G-protein signaling molecules. Our assessment of synaptic function in Ric mutants, combined with the homologies of some Ric mutants to presynaptic proteins, suggests that the analysis of Ric genes will continue to yield insights into the regulation and functioning of synapses.

A deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is replication defective due to a failure in autoregulation.

Human cytomegalovirus (CMV) replication begins with the expression of two regulatory proteins, IE1(491aa) and IE2(579aa), produced from differentially spliced transcripts under control of the ie1/ie2 promoter-enhancer. A deletion mutation removing all 406 IE1(491aa)-specific amino acids was engineered into the viral genome and this mutant (RC303 delta Acc) was propagated on an IE1(491aa)-expressing human fibroblast cell line (ihfie1.3). RC303 delta Acc failed to replicate on normal human fibroblasts at low multiplicities of infection (mois). At mois > 3 plaque-forming units per cell, virus replication and production of progeny were comparable to wild type. However, at mois between 0.01 and 1, mutant virus replicated slowly on normal fibroblasts, a pattern that suggested initiation of productive infection required multiple hits. Replication of RC303 delta Acc correlated with the ability to express IE2(579aa), consistent with a role for IE1(491aa) in positive autoregulation of the ie1/ie2 promoter-enhancer and with data suggesting that virion transactivators compensate for the lack of IE1(491aa) under high moi conditions. ie1-deficient CMV should be completely avirulent, suggesting its utility as a gene therapy vector for hematopoietic progenitors that are normal sites of CMV latency.

Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines.

Stable mammalian cell lines harboring a synthetic bovine opsin gene have been derived from the suspension-adapted HEK293 cell line. The opsin gene is under the control of the immediate-early cytomegalovirus promoter/enhancer in an expression vector that also contains a selectable marker (Neo) governed by a relatively weak promoter. The cell lines expressing the opsin gene at high levels are selected by growth in the presence of high concentrations of the antibiotic geneticin. Under the conditions used for cell growth in suspension, opsin is produced at saturated culture levels of more than 2 mg/liter. After reconstitution with 11-cis-retinal, rhodopsin is purified to homogeneity in a single step by immunoaffinity column chromatography. Rhodopsin thus prepared (> 90% recovery at concentrations of up to 15 microM) is indistinguishable from rhodopsin purified from bovine rod outer segments by the following criteria: (i) UV/Vis absorption spectra in the dark and after photobleaching and the rate of metarhodopsin II decay, (ii) initial rates of transducin activation, and (iii) the rate of phosphorylation by rhodopsin kinase. Although mammalian cell opsin migrates slower than rod outer segment opsin on SDS/polyacrylamide gels, presumably due to a different N-glycosylation pattern, their mobilities after deglycosylation are identical. This method has enabled the preparation of several site-specific mutants of bovine opsin in comparable amounts.