Showing papers on "Mutant published in 1988"

Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes

Abstract: Gene targeting—homologous recombination of DNA sequences residing in the chromosome with newly introduced DNA sequences—in mouse embryo-derived stem cells promises to provide a means to generate mice of any desired genotype. We describe a positive and negative selection procedure that enriches 2,000-fold for those cells that contain a targeted mutation. The procedure was applied to the isolation of hprt− and int-2− mutants, but it should be applicable to any gene

The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus.

Abstract: Mice carrying mutations at the W locus located on chromosome 5 are characterized by severe macrocytic anaemia, lack of hair pigmentation and sterility1. Mutations at this locus appear to affect the proliferation and/or migration of cells during early embryogenesis and result in an intrinsic defect in the haematopoietic stem cell hierarchy1,2. An understanding of the molecular basis of the complex and pleiotropic phenotype in W mutant mice would thus provide insights into the important developmental processes of gametogenesis, melanogenesis and haematopoiesis. Here we show that the mouse mutant W19H has a deletion of the c-kit proto-oncogene. Interspecific backcross analysis demonstrates that the W locus is very tightly linked to c-kit and that the two loci cannot be segregated at this level of analysis, c-kit is the cellular homologue of the oncogene v-kit of the HZ4 feline sarcoma virus3 and encodes a transmembrane protein tyrosine kinase receptor that is structurally similar to the receptors for colony-stimulating factor-1 (CSF-1) and platelet derived growth factor4,5. The co-localization of c-kit with W provides a molecular entry into this important region of the mouse genome. In addition, these observations provide the first example of a germ-line mutation in a mammalian proto-oncogene and implicate the c-kit gene as a candidate for the W locus.

Active human immunodeficiency virus protease is required for viral infectivity.

Abstract: Retroviral proteins are synthesized as polyprotein precursors that undergo proteolytic cleavages to yield the mature viral proteins. The role of the human immunodeficiency virus (HIV) protease in the viral replication cycle was examined by use of a site-directed mutation in the protease gene. The HIV protease gene product was expressed in Escherichia coli and observed to cleave HIV gag p55 to gag p24 and gag p17 in vitro. Substitution of aspartic acid residue 25 (Asp-25) of this protein with an asparagine residue did not affect the expression of the protein, but it eliminated detectable in vitro proteolytic activity against HIV gag p55. A mutant HIV provirus was constructed that contained the Asn-25 mutation within the protease gene. SW480 human colon carcinoma cells transfected with the Asn-25 mutant proviral DNA produced virions that contained gag p55 but not gag p24, whereas virions from cells transfected with the wild-type DNA contained both gag p55 and gag p24. The mutant virions were not able to infect MT-4 lymphoid cells. In contrast, these cells were highly sensitive to infection by the wild-type virions. These results demonstrate that the HIV protease is an essential viral enzyme and, consequently, an attractive target for anti-HIV drugs.

Chloroplast transformation in Chlamydomonas with high velocity microprojectiles.

Abstract: Bombardment of three mutants of the chloroplast atpB gene of Chlamydomonas reinhardtii with high-velocity tungsten microprojectiles that were coated with cloned chloroplast DNA carrying the wild-type gene permanently restored the photosynthetic capacity of the algae. In most transformants of one of the mutants, a fragment with a 2.5-kilobase deletion was restored to normal size by a homologous replacement event; in about 25 percent of the transformants the restored restriction fragment was 50 to 100 base pairs smaller or larger than that of wild type. About one-fourth of the transformants of this mutant contained unintegrated donor plasmid when first examined. This plasmid persisted in four different transformants after 65 cell generations of continuous liquid culture but was lost from all transformants maintained on plates of selective medium. The restored wild-type atpB gene remains in all transformants as an integral part of the chloroplast genome and is expressed and inherited normally.

The origin of mutants

Abstract: Nucleic acids are replicated with conspicuous fidelity. Infrequently, however, they undergo changes in sequence, and this process of change (mutation) generates the variability that allows evolution. As the result of studies of bacterial variation, it is now widely believed that mutations arise continuously and without any consideration for their utility. In this paper, we briefly review the source of this idea and then describe some experiments suggesting that cells may have mechanisms for choosing which mutations will occur.

Insensitivity to Ethylene Conferred by a Dominant Mutation in Arabidopsis thaliana

Abstract: Ethylene influences a number of developmental processes and responses to stress in higher plants. The molecular basis for the action of ethylene was investigated in mutants of Arabidopsis thaliana that have altered responses to ethylene. One mutant line, which has a dominant mutation at a locus designated etr, lacks a number of responses to ethylene that are present in the wild-type plant. These include inhibition of cell elongation, promotion of seed germination, enhancement of peroxidase activity, acceleration of leaf senescence, and feedback suppression of ethylene synthesis by ethylene. These diverse responses, which occur in different tissues of Arabidopsis, appear to share some common element in their transduction pathways—for example, a single receptor for ethylene. Results of ethylene binding experiments in vivo indicate that this receptor may be affected by the etr mutation.

Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases

Abstract: Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature sensitive for vegetative cell growth. This temperature-sensitive phenotype has been shown to be recessive and to cosegregate with the vacuolar protein-sorting defect in each case. Tetrad analysis showed that vpt3 mapped to the right arm of chromosome XV and that vpt15 mapped to the right arm of chromosome II. Intercrosses with other mutants that exhibited defects in vacuolar protein sorting or function (vpl, sec, pep, and end mutants) revealed several overlaps among these different sets of genes. Together, these data indicate that more than 50 gene products are involved, directly or indirectly, in the process of vacuolar protein sorting.

Cell binding function of E-cadherin is regulated by the cytoplasmic domain.

Abstract: Cadherins are a family of transmembrane glycoproteins responsible for Ca2+-dependent cell-cell adhesion. Their amino acid sequences are highly conserved in the cytoplasmic domain. To study the role of the cytoplasmic domain in the function of cadherins, we constructed expression vectors with cDNAs encoding the deletion mutants of E-cadherin polypeptides, in which the carboxy terminus was truncated at various lengths. These vectors were introduced into L cells by transfection, and cell lines expressing the mutant E-cadherin molecules were isolated. In all transfectants obtained, the extracellular domain of the mutant E-cadherins was exposed on the cell surface, and had normal Ca2+-sensitivity and molecular size. However, these cells did not show any Ca2+-dependent aggregation, indicating that the mutant molecules cannot mediate cell-cell binding. The mutant E-cadherin molecules could be released from cells by nonionic detergents, whereas a fraction of normal E-cadherin molecules could not be extracted with the detergent and appeared to be anchored to the cytoskeleton at cell-cell junctions. These results suggest that the cytoplasmic domain regulates the cell-cell binding function of the extracellular domain of E-cadherin, possibly through interaction with some cytoskeletal components.

Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

Abstract: Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.

In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity.

Abstract: Site-specific mutagenesis was used to introduce amino acid substitutions at the asparagine codons of four conserved potential N-linked glycosylation sites within the gp120 envelope protein of human immunodeficiency virus (HIV). One of these alterations resulted in the production of noninfectious virus particles. The amino acid substitution did not interfere with the synthesis, processing, and stability of the env gene polypeptides gp120 and gp41 or the binding of gp120 to its cellular receptor, the CD4 (T4) molecule. Vaccinia virus recombinants containing wild-type or mutant HIV env genes readily induced syncytia in CD4+ HeLa cells. These results suggest that alterations involving the second conserved domain of the HIV gp120 may interfere with an essential early step in the virus replication cycle other than binding to the CD4 receptor. In long-term cocultures of a T4+ lymphocyte cell line and colon carcinoma cells producing the mutant virus, revertant infectious virions were detected. Molecular characterization of two revertant proviral clones revealed the presence of the original mutation as well as a compensatory amino acid change in another region of HIV gp120.

The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog.

Abstract: WHI1-1 is a dominant mutation that reduces cell volume by allowing cells to commit to division at abnormally small sizes, shortening the G1 phase of the cell cycle. The gene was cloned, and dosage studies indicated that the normal gene activated commitment to division in a dose-dependent manner, and that the mutant gene had a hyperactive but qualitatively similar function. Mild over-expression of the mutant gene eliminated G1 phase, apparently entirely relaxing the normal G1 size control, but revealing hitherto cryptic controls. Sequence analysis showed that the hyperactivity of the mutant was caused by the loss of the C-terminal third of the wild-type protein. This portion of the protein contained PEST regions, which may be signals for protein degradation. The WHI1 protein had sequence similarity to clam cyclin A, to sea urchin cyclin and to Schizosaccharomyces pombe cdc13, a cyclin homolog. Since cyclins are inducers of mitosis, WHI1 may be a direct regulator of commitment to division. A probable accessory function of the WHI1 activator is to assist recovery from alpha factor arrest; WHI1-1 mutant cells could not be permanently arrested by pheromone, consistent with a hyperactivation of division.

Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli.

Abstract: Eight mutants of Alcaligenes eutrophus defective in the intracellular accumulation of poly-beta-hydroxybutyric acid (PHB) were isolated after transposon Tn5 mutagenesis with the suicide vector pSUP5011. EcoRI fragments which harbor Tn5-mob were isolated from pHC79 cosmid gene banks. One of them, PPT1, was used as a probe to detect the intact 12.5-kilobase-pair EcoRI fragment PP1 in a lambda L47 gene bank of A. eutrophus genomic DNA. In six of these mutants (PSI, API, GPI, GPIV, GPV, and GPVI) the insertion of Tn5-mob was physically mapped within a region of approximately 1.2 kilobase pairs in PP1; in mutant API, cointegration of vector DNA has occurred. In two other mutants (GPII and GPIII), most probably only the insertion element had inserted into PP1. All PHB-negative mutants were completely impaired in the formation of active PHB synthase, which was measured by a radiometric assay. In addition, activities of beta-ketothiolase and of NADPH-dependent acetoacetyl coenzyme A (acetoacetyl-CoA) reductase were diminished, whereas the activity of NADPH-dependent acetoacetyl-CoA reductase was unaffected. In all PHB-negative mutants the ability to accumulate PHB was restored upon complementation in trans with PP1. The PHB-synthetic pathway of A. eutrophus was heterologously expressed in Escherichia coli. Recombinant strains of E. coli JM83 and K-12, which harbor pUC9-1::PP1, pSUP202::PP1, or pVK101::PP1, accumulated PHB up to 30% of the cellular dry weight. Crude extracts of these cells had significant activities of the enzymes PHB synthase, beta-ketothiolase, and NADPH-dependent acetoacetyl-CoA reductase. Therefore, PP1 most probably encodes all three genes of the PHB-synthetic pathway in A. eutrophus. In addition to PHB-negative mutants, we isolated mutants which accumulate PHB at a much lower rate than the wild type does. These PHB-leaky mutants exhibited activities of all three PHB-synthetic enzymes; Tn5-mob had not inserted into PP1, and the phenotype of the wild type could not be restored with fragment PP1. The rationale for this mutant type remains unknown.

DNA topoisomerase-targeting antitumor drugs can be studied in yeast

Abstract: The antitumor drugs camptothecin and an anilinoacridine, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA), which act on DNA topoisomerase I and II, respectively, are shown to inhibit the growth of Saccharomyces cerevisiae mutants selected for their permeability to other inhibitors. In addition to growth inhibition, these drugs induce high levels of homologous recombination and induce the expression of a DNA damage-inducible gene DIN3. Cytotoxicity of the drugs is more pronounced in strains that also carry a rad52 mutation. An analog of mAMSA), which is ineffective as an inhibitor of DNA topoisomerase II in mammalian cells, is also ineffective in eliciting physiological responses in these yeast strains. The physiological effects of camptothecin, but not those of mAMSA, disappear if the TOP1 gene encoding DNA topoisomerase I is disrupted. This shows that DNA topoisomerase I is the sole target of camptothecin cytotoxicity and illustrates that a nonessential enzyme can nevertheless be the target for a cytotoxic drug.

arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways.

Abstract: In Escherichia coli the levels of numerous enzymes associated with aerobic metabolism are decreased during anaerobic growth. In an arcA mutant the anaerobic levels of these enzymes are increased. The enzymes, which are encoded by different regulons, include members that belong to the tricarboxylic acid cycle, the glyoxylate shunt, the pathway for fatty acid degradation, several dehydrogenases of the flavoprotein class, and the cytochrome o oxidase complex. Transductional crosses placed the arcA gene near min O on the chromosomal map. Complementation tests showed that the arcA gene corresponded to the dye gene, which is also known as fexA, msp, seg, or sfrA because of various phenotypic properties [Bachmann, B. (1983) Microbiol. Rev. 47, 180-230]. A dye-deletion mutant was derepressed in the aerobic enzyme system. The term modulon is proposed to describe a set of regulons that are subject to a common transcriptional control.

Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant.

Abstract: Herpes simplex virus (HSV) encodes a ribonucleotide reductase consisting of two subunits (140 and 38 kilodaltons) whose genes map to coordinates 0.56 to 0.60 on the viral genome. Host cell lines containing the HpaI F fragment which includes the reductase subunit genes of HSV type 1 strain KOS (coordinates 0.535 to 0.620) were generated. Transfection of these cells with a plasmid containing the immediate-early ICP0 gene resulted in the expression of ICP6; interestingly, ICP4 plasmids failed to induce expression, indicating an unusual pattern of ICP6 regulation. One such cell line (D14) was used to isolate a mutant with the structural gene of lacZ inserted into the ICP6 gene such that the lacZ gene is read in frame with the N-terminal region of ICP6. This mutant generated a protein containing 434 amino acids (38%) of the N terminus of ICP6 fused to beta-galactosidase under control of the endogenous ICP6 promoter. Screening for virus recombinants was greatly facilitated by staining virus plaques with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside (X-gal). Enzyme assays of infected BHK cells indicated that the mutant is incapable of inducing viral ribonucleotide reductase activity. Surprisingly, although plaque size was greatly reduced, mutant virus yield was reduced only four- to fivefold compared with that of the wild type grown in exponentially growing Vero cells. Mutant virus plaque size, yields, and ability to synthesize viral DNA were more severely compromised in serum-starved cells as compared with the wild type grown under the same condition. Although our evidence suggests that the HSV type 1 ribonucleotide reductase is not required for virus growth and DNA replication in dividing cells, it may be required for growth in nondividing cells.

An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins

Abstract: A fusion between tsr (encoding the inner membrane protein Tsr) and phoA (encoding the periplasmic protein alkaline phosphatase, AP) generates a membrane-bound hybrid protein (Tsr-AP 2) with AP enzymatic activity. The hybrid protein is proteolytically unstable and is broken down to yield a smaller, soluble species with AP activity. We devised a genetic screen to distinguish between cells containing only membrane-bound AP and those containing soluble AP. The screen depends on diffusion of soluble AP away from cells with a leaky outer membrane to produce a halo of AP activity around colonies on solid growth medium. Several mutants lacking this halo show reduced degradation of Tsr-AP 2. One mutant is also defective in breakdown of five other abnormal periplasmic proteins but not of two cytoplasmic proteins. The mutation in this strain, degP4::Tn5, defines a locus distinct from previously identified loci that affect protein stability or protease activities. This strain may be useful for preventing the breakdown of unstable foreign proteins in Escherichia coli.

Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis.

Abstract: Summary Sporulation begins coincidentally with the expression of several stationary-phase-associated gene products during the transition state of a culture from exponential to stationary phase. Mutations in the stage 0 sporulation genes prevent the expression of these gene products in addition to blocking sporulation. Suppressor mutations in the abrB gene, in a spo0 background, restore stationary-phase-associated gene expression but not sporulation. The nature of the abrB gene product was investigated by isolating and sequencing the abrB gene. The abrB gene coded for a 96-amino-acid protein (molecular weight 10773) and contained a helix-turn-helix structure common to DNA binding proteins. Analysis of expression of the abrB gene using lacZ transcription fusions and direct measurement of mRNA content by hybridization showed that the spo0A gene repressed transcription of the abrB gene. Primer extension analysis of abrB gene mRNA revealed two initiation sites. The downstream site was dramatically repressed in spo0A+ strains, while the upstream site appeared not to be regulated by spo0A. Five abrB mutant alleles were cloned and sequenced. One mutation, abrB 4, resided within the structural gene and continued to overexpress abrB messenger RNA from both promoters. A promoter mutation, abrB 15, reduced transcription from the downstream promoter but not the upstream promoter. Thus, the phenotype of abrB mutations results from inactivation of the abrB gene product or by prevention of its overexpression. The results suggest that the abrB gene codes for a regulator which controls several genes whose products are normally produced during the transition phase between active growth and sporulation. The level of this regulator is, in turn, controlled by the spo0A gene. The pleiotropic phenotypes of spo0A mutants result from uncontrolled overexpression of the abrB regulator.

Herpes simplex virus virion host shutoff function.

Abstract: Herpes simplex virus (HSV) virions contain one or more functions which mediate the shutoff of host protein synthesis and the degradation of host mRNA. HSV type 1 (HSV-1) mutants deficient in the virion shutoff of host protein synthesis (vhs mutants) were isolated and were found to be defective in their ability to degrade host mRNA. Furthermore, it was found that viral mRNAs in cells infected with the vhs 1 mutant have a significantly longer functional half-life than viral mRNAs in wild-type virus-infected cells. In the present study we have mapped the vhs1 mutation affecting the virion shutoff of host protein synthesis to a 265-base-pair NruI-XmaIII fragment spanning map coordinates 0.604 to 0.606 of the HSV-1 genome. The mutation(s) affecting the functional half-lives of host mRNA as well as the alpha (immediate-early), beta (early), and gamma (late) viral mRNAs were also mapped within this 265-base-pair fragment. Thus, the shutoff of host protein synthesis is most likely mediated by the same function which decreases the half-life of viral mRNA. The shorter half-life of infected-cell mRNAs may allow a more rapid modulation of viral gene expression in response to changes in the transcription of viral genes. Interestingly, the vhs1 mutation of HSV-1 maps within a region which overlaps the Bg/II-N sequences of HSV-2 DNA shown previously to transform cells in culture. The possible relationship between the transformation and host shutoff functions are discussed.

Nitrate reduction mutants of fusarium moniliforme (gibberella fujikuroi).

Abstract: Twelve strains of Fusarium moniliforme were examined for their ability to sector spontaneously on toxic chlorate medium. All strains sectored frequently; 91% of over 1200 colonies examined formed chlorate-resistant, mutant sectors. Most of these mutants had lesions in the nitrate reduction pathway and were unable to utilize nitrate (nit mutants). nit mutations occurred in seven loci: a structural gene for nitrate reductase (nit1), a regulatory gene specific for the nitrate reduction pathway (nit3), and five genes controlling the production of a molybdenum-containing cofactor that is necessary for nitrate reductase activity (nit2, nit4, nit5, nit6, nit7). No mutations affecting nitrite reductase or a major nitrogen regulatory locus were found among over 1000 nit mutants. Mutations of nit1 were recovered most frequently (39-66%, depending on the strain) followed by nit3 mutations (23-42%). The frequency of isolation of each mutant type could be altered, however, by changing the source of nitrogen in the chlorate medium. We concluded that genetic control of nitrate reduction in F. moniliforme is similar to that in Aspergillus and Neurospora, but that the overall regulation of nitrogen metabolism may be different.

Inhibition of phagocytosis in Yersinia pseudotuberculosis: a virulence plasmid-encoded ability involving the Yop2b protein.

Abstract: Virulence plasmid-containing cells of Yersinia pseudotuberculosis had the ability to inhibit phagocytosis by mouse peritoneal macrophages cultured in vitro, but cells of its plasmid-cured derivative did not. Inhibition was most pronounced when the pathogen was incubated under Ca2+-deficient conditions, which allowed a high level of expression of outer membrane proteins (Yops). The addition of 2.5 mM Ca2+ to the growth medium reduced the degree of inhibition by the pathogen, but it was still significantly higher than that of the plasmid-cured strain. An avirulent mutant strain, from which the entire yopH gene was deleted, was impaired in its phagocytosis inhibition ability. This mutant could be trans-complemented by the yopH+ gene back to the wild-type phenotype with respect to virulence, as well as the ability to inhibit phagocytosis, demonstrating that the ability to inhibit phagocytosis is an important virulence function. The mutant strain was still cytotoxic for HeLa cells, indicating that inhibition of phagocytosis can be genetically separated from the ability to cause a cytotoxic effect. Images

Identification of the human c-myc protein nuclear translocation signal.

Abstract: We identified and characterized two regions of the human c-myc protein that target proteins into the nucleus. Using mutant c-myc proteins and proteins that fuse portions of c-myc to chicken muscle pyruvate kinase, we found that residues 320 to 328 (PAAKRVKLD; peptide M1) induced complete nuclear localization, and their removal from c-myc resulted in mutant proteins that distributed in both the nucleus and cytoplasm but retained rat embryo cell cotransforming activity. Residues 364 to 374 (RQRRNELKRSP; peptide M2) induced only partial nuclear targeting, and their removal from c-myc resulted in mutant proteins that remained nuclear but were cotransformationally inactive. We conjugated synthetic peptides containing M1 or M2 to human serum albumin and microinjected the conjugate into the cytoplasm of Vero cells. The peptide containing M1 caused rapid and complete nuclear accumulation, whereas that containing M2 caused slower and only partial nuclear localization. Thus, M1 functions as the nuclear localization signal of c-myc, and M2 serves some other and essential function.

Isolation and characterization of a starchless mutant of Arabidopsis thaliana (L. ) Heynh lacking ADPglucose pyrophosphorylase activity

Abstract: A mutant of Arabidopsis thaliana lacking ADPglucose pyrophosphorylase activity (EC 2.7.7.27) was isolated (from a mutagenized population of plants) by screening for the absence of leaf starch. The mutant grows as vigorously as the wild type in continuous light but more slowly than the wild type in a 12 hours light/12 hours dark photoperiod. Genetic analysis showed that the deficiency of both starch and ADPglucose pyrophosphorylase activity were attributable to a single, nuclear, recessive mutation at a locus designated adg1. The absence of starch in the mutant demonstrates that starch synthesis in the chloroplast is entirely dependent on a pathway involving ADPglucose pyrophosphorylase. Analysis of leaf extracts by two-dimensional polyacrylamide gel electrophoresis followed by Western blotting experiments using antibodies specific for spinach ADPglucose pyrophosphorylase showed that two proteins, present in the wild type, were absent from the mutant. The heterozygous F1 progeny of a cross between the mutant and wild type had a specific activity of ADPglucose pyrophosphorylase indistinguishable from the wild type. These observations suggest that the mutation in the adg1 gene in TL25 might affect a regulatory locus.