Showing papers on "Mutant published in 1986"

A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma

Abstract: The genomes of various tumour cells contain mutant oncogenes that act dominantly, in that their effects can be observed when they are introduced into non-malignant cells. There is evidence for another class of oncogenes, in which tumour-predisposing mutations are recessive to wild-type alleles. Retinoblastoma is a prototype biological model for the study of such recessive oncogenes. This malignant tumour, which arises in the eyes of children, can be explained as the result of two distinct genetic changes, each causing loss of function of one of the two homologous copies at a single genetic locus, Rb, assigned to the q14 band of human chromosome 13. Mutations affecting this locus may be inherited from a parent, may arise during gametogenesis or may occur somatically. Those who inherit a mutant allele at this locus have a high incidence of non-ocular, second tumours, almost half of which are osteosarcomas believed to be caused by the same mutation. Here we describe the isolation of a complementary DNA segment that detects a chromosomal segment having the properties of the gene at this locus. The gene is expressed in many tumour types, but no RNA transcript has been found in retinoblastomas and osteosarcomas. The cDNA fragment detects a locus spanning at least 70 kilobases (kb) in human chromosome band 13q14, all or part of which is frequently deleted in retinoblastomas and osteosarcomas.

Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life?

Abstract: Mu transposons carrying the chloramphenicol resistance marker have been inserted into the cloned Escherichia coli genes sodA and sodB coding for manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) respectively, creating mutations and gene fusions. The mutated sodA or sodB genes were introduced into the bacterial chromosome by allelic exchange. The resulting mutants were shown to lack the corresponding SOD by activity measurements and immunoblot analysis. Aerobically, in rich medium, the absence of FeSOD or MnSOD had no major effect on growth or sensitivity to the superoxide generator, paraquat. In minimal medium aerobic growth was not affected, but the sensitivity to paraquat was increased, especially in the sodA mutant. A sodA sodB double mutant completely devoid of SOD was also obtained. It was able to grow aerobically in rich medium, its catalase level was unaffected and it was highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose medium. Growth could be restored by removing oxygen, by providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that the total absence of SOD in E. coli creates a conditional sensitivity to oxygen.

Sulfonylurea-resistant mutants of Arabidopsis thaliana

Abstract: Chlorsulfuron-resistant mutants of Arabidopsis thaliana were isolated by screening for growth of seedlings in the presence of the herbicide. Both whole plants and derived tissue cultures were resistant to concentrations of the herbicide approximately 300-fold higher than that required to prevent growth of the wild-type. The resistance is due to a single dominant nuclear mutation at a locus designated csr which has been genetically mapped to chromosome-3. Acetohydroxy acid synthase activity in extracts from chlorsulfuron-resistant plants was much less-susceptible to inhibition by chlorsulfuron and a structurally related inhibitor than the activity in wild-type extracts. This suggests that the csr locus is the structural gene for acetohydroxy acid synthase.

Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

Abstract: We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.

Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid.

Abstract: Biosynthesis of HLA class I antigens has been studied in a variant B-LCLxT-LCL hybrid, 174XCEM.T2. This cell line encodes HLA-A2 and -B5, but expresses only small amounts of A2 antigen and undetectable B5 antigen at the cell surface due to a mutation inactivating a trans-acting regulatory gene encoded within the class II region of the human major histocompatibility complex. Northern blot analysis with HLA-A- and HLA-B-specific probes shows that 174XCEM.T2 synthesizes quantities of A and B locus mRNA comparable with its class I antigen-positive parent cell line. Immune precipitation studies indicate that 174XCEM.T2 synthesizes normal HLA heavy chains and beta 2-microglobulin which fail to form dimers. The heavy chains are N-glycosylated normally, but processing of the glycan to the complex form does not occur. In addition, free heavy chains in this cell line are not phosphorylated. Thus, the majority of class I heavy chains in 174XCEM.T2 do not combine with beta 2-microglobulin, and are not processed or transported to the cell surface. As both subunits are synthesized in normal amounts, we propose that an additional molecule absent from 174XCEM.T2 and encoded by an HLA-linked gene is necessary for efficient assembly of class I antigen subunits.

Isolation and characterization of a herpes simplex virus type 1 mutant containing a deletion within the gene encoding the immediate early polypeptide Vmw110

Abstract: Transfection experiments with plasmids containing immediate early (IE) genes of herpes simplex virus type 1 (HSV-1) have previously demonstrated a role for the IE polypeptide Vmw110 (ICP0) in stimulating expression from plasmid-encoded early gene promoters. To gain further insights into the function of Vmw110 we isolated a deletion mutant specifying a truncated form of the polypeptide which had been shown to be inactive in transfection assays. This mutant, dl1403, contained a 2 kb deletion within both the TRL and IRL copies of the Vmw110 gene, and encoded a polypeptide consisting of the original N-terminal 105 amino acids followed by 56 amino acids specified by a reading frame not used by Vmw110. dl1403 was able to replicate and produce plaques on baby hamster kidney (BHK) cells but the yield of infectious virus was 20- to 100-fold lower than obtained with wild-type HSV-1. Surprisingly, comparison of polypeptide synthesis, DNA replication and DNA encapsidation in cells infected with 5 p.f.u./cell dl1403 or wild-type HSV-1 revealed no significant differences. In addition similar numbers of particles were produced in cells infected with the two viruses, resulting in stocks of dl1403 exhibiting significantly higher particle/p.f.u. ratios. The efficiency of plaquing of dl1403 was greatly reduced in Vero and human foetal lung cells compared with BHK cells, but following infection with 5 p.f.u./cell similar yields of infectious virus were obtained from all three cell lines. Marker rescue experiments verified that the reduced yield of dl1403 in BHK cells was a consequence of the deletion within the Vmw110 gene. The results suggest that the effect of this deletion is manifest primarily at low multiplicities of infection and can be largely overcome by increasing the virus dose.

PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors.

Abstract: The proteinase A structural gene of Saccharomyces cerevisiae was cloned by using an immunological screening procedure that allows detection of yeast cells which are aberrantly secreting vacuolar proteins (J. H. Rothman, C. P. Hunter, L. A. Valls, and T. H. Stevens, Proc. Natl. Acad. Sci. USA, 83:3248-3252, 1986). A second cloned gene was obtained on a multicopy plasmid by complementation of a pep4-3 mutation. The nucleotide sequences of these two genes were determined independently and were found to be identical. The predicted amino acid sequence of the cloned gene suggests that proteinase A is synthesized as a 405-amino-acid precursor which is proteolytically converted to the 329-amino-acid mature enzyme. Proteinase A shows substantial homology to mammalian aspartyl proteases, such as pepsin, renin, and cathepsin D. The similarities may reflect not only analogous functions but also similar processing and intracellular targeting mechanisms for the two proteins. The cloned proteinase A structural gene, even when it is carried on a single-copy plasmid, complements the deficiency in several vacuolar hydrolase activities that is observed in a pep4 mutant. A strain carrying a deletion in the genomic copy of the gene fails to complement a pep4 mutant of the opposite mating type. Genetic linkage data demonstrate that integrated copies of the cloned proteinase A structural gene map to the PEP4 locus. Thus, the PEP4 gene encodes a vacuolar aspartyl protease, proteinase A, that is required for the in vivo processing of a number of vacuolar zymogens.

Endonuclease IV (nfo) mutant of Escherichia coli.

Abstract: A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested.

The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs.

Abstract: The adenovirus type 5 mutant H5dl338 lacks 524 base pairs within early region 1B. The mutation removed a portion of the region encoding the related E1B-55K and -17K polypeptides but did not disturb the E1B-21K coding region. The virus can be propagated in 293 cells which contain and express the adenovirus type 5 E1A and E1B regions, but it is defective for growth in HeLa cells, in which its final yield is reduced about 100-fold compared with the wild-type virus. The mutant also fails to transform rat cells at normal efficiency. The site of the dl338 defect was studied in HeLa cells. Early gene expression and DNA replication appeared normal. Late after infection, mRNAs coded by the major late transcription unit accumulated to reduced levels. At a time when transcription rates and steady-state nuclear RNA species were normal, the rate at which late mRNA accumulated in the cytoplasm was markedly reduced. Furthermore, in contrast to the case with the wild type, transport and accumulation of cellular mRNAs continued late after infection with dl338. Thus, the E1B product appears to facilitate transport and accumulation of viral mRNAs late after infection while blocking the same processes for cellular mRNAs.

Effect of gamma rays at the dihydrofolate reductase locus: deletions and inversions

Abstract: A series 11 gamma-ray-induced mutants at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary cells has been examined for the types of DNA sequence change brought about by this form of ionizing radiation. All 11 mutants were found to have suffered major structural changes affecting the dhfr gene. In eight of the mutants, all or part of the dhfr gene has been deleted. The extent of these deletions was examined in seven of these mutants and, for comparison, in two deletion mutants that were induced by UV irradiation. For this purpose, probes from an overlapping set of cosmids that span 210 kb of DNA in this region were used. Three of seven gamma-ray-induced mutants and one UV-induced mutant were shown to have deleted the entire 210-kb region. In the remaining mutants, endpoints ranging from within the dhfr gene to 100 kb downstream were observed. No upstream endpoints were detected, so that an upper limit on the size of these large deletions could not be assigned. Three of the 11 gamma-ray-induced mutants contained an interruption in the dhfr gene without any detectable loss of sequence. Restriction analysis of these interrupted mutants showed that at least 8-14 kb of "foreign" DNA sequence became joined to the gene at the point of disruption. Cytogenetic analysis of these mutants showed that in two cases an inversion of the banding pattern on chromosome Z-2 had taken place. The inverted dhfr mutants contain very low amounts of dhfr RNA sequences, and the 5' end of an inversion mutant gene exhibits the same pattern of DNA methylation and DNase I-hypersensitivity as the wild-type gene. Our results suggest that ionizing radiation causes primarily, if not exclusively, large deletions and inversions in mammalian cells.

Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp.

Abstract: Genetic transformation of auxotrophs of the extreme thermophile Thermus thermophilus HB27 to prototrophy was obtained at high frequencies of 10(-2) to 10(-1) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent wild-type strain. The transformation frequency was proportional to the DNA concentration from 10 pg/ml to 100 ng/ml. T. thermophilus HB27 cells did not require chemical treatment to induce competence, although optimal transformation was obtained by the addition of a divalent cation (Ca2+ or Mg2+). Competence was maintained throughout the growth phase, with the highest transformation frequencies at pH 6 to 9 and at 70 degrees C. T. thermophilus HB27 and four other typical Thermus strains, T. thermophilus HB8, T. flavus AT62, T. caldophilus GK24, and T. aquaticus YT1, were also transformed to streptomycin resistance by DNA from their own spontaneous streptomycin-resistant mutants. A cryptic plasmid, pTT8, from T. thermophilus HB8 was introduced into T. thermophilus HB27 Pro- at a frequency of 10(-2).

The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases.

Abstract: pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.

Myristylation site in Pr65gag is essential for virus particle formation by moloney murine leukemia virus

Abstract: It was previously reported that the gag proteins of mammalian type C retroviruses are modified by the addition of myristate to the N-terminal glycine residue. We have performed oligonucleotide-directed mutagenesis to change this glycine codon in the Moloney murine leukemia virus genome to an alanine codon and also to specifically delete the glycine codon. Upon transfection into mammalian cells, these mutant genomes direct the synthesis of gag proteins, but these proteins are not myristylated. The mutants do not form virus particles or any recognizable virus-specific structures visible in thin sections with the electron microscope. Further, the mutant gag proteins appear to remain in the cytosol, whereas the wild type is found principally in particulate fractions of the cell. The results are consistent with the theory that myristate is required for the association of the gag protein with the plasma membrane and that this association is necessary for virus assembly.

Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis.

Abstract: By examining cytological phenotypes of 587 temperature-sensitive mutants of the fission yeast Schizosaccharomyces pombe, we obtained 18 mutants which cause cell division in the absence of nuclear division. By genetic analyses, these novel nuclear division arrest mutants can be classified into nine complementation groups (designated cut1 – cut9). The cytological phenotype of cut mutants is similar but not identical to that of DNA topoisomerase II mutants (top2). The cut1+ gene was cloned by transformation and shown to complement cut2 as well as cut1, indicating a functional relationship between the two genes. The cut genes are required for nuclear division, but their mutant phenotypes differ from most of the previously identified mutants which block nuclear division and also the subsequent cytokinesis. Fluorescence microscopy indicates that the mitotic chromosomes formed in cut mutant cells are abnormal and fail to separate properly. We suggest that cut mutations, like top2, block mitotic chromosome formation and concomitantly nuclear division, but that cytokinesis proceeds independently of the defects in nuclear division, demonstrating uncoordinated mitotic pathways. A novel mutant nuc1 is also described which shows a cytological phenotype similar to the double mutant of DNA topoisomerases I and II but contains normal levels of both DNA topoisomerase activities.

Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae

Abstract: A gene, PDE2, has been cloned from the yeast Saccharomyces cerevisiae that, when present in high copy, reverses the phenotypic effects of RAS2Val19, a mutant form of the RAS2 gene that renders yeast cells sensitive to heat shock and starvation. It has previously been shown that the RAS proteins are potent activators of yeast adenylate cyclase. We report here that PDE2 encodes a high-affinity cAMP phosphodiesterase that shares sequence homology with animal cell phosphodiesterases. These results therefore imply that the effects of RAS2Val19 are mediated through its changes in cAMP concentration.

Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor

Abstract: Haploid yeast cells of the a mating type secrete a peptide pheromone, a factor, which acts on cells of the alpha mating type to prepare them for conjugation. We show that the STE3 gene, which is required for mating only by alpha cells and is transcribed only in alpha cells, likely encodes a cell-surface receptor for a factor. This view is based on three findings. First, wild-type Ste3 product is required for response to the pheromone: mutants with any one of five different ste3 mutations are unresponsive to a factor. Second, a hybrid Ste3-beta-galactosidase protein encoded by a STE3-lacZ gene fusion fractionates to the particulate fraction of yeast cell extracts, suggesting that Ste3 is a membrane protein. Finally, the DNA sequence of STE3, which we report here, encodes a protein of 470 amino acid residues that contains seven distinct hydrophobic segments of sufficient length to span a lipid bilayer.

Murine Major Histocompatibility Complex Class-I Mutants: Molecular Analysis and Structure-Function Implications

Abstract: The class-I mutants have provided a model system for understanding the generation of diversity of the genes encoding the histocompatibility molecules K, D, and L, and the relationship of their structure to function. The complex nature of the alterations found in Kb molecules from mutant mice has been documented at the nucleic acid level for eight mutants. The clustered changes in the mutant genes are consistent with the hypothesis that genetic recombination between class-I genes generates the Kb mutants. Techniques using synthetic oligonucleotide probes to mutant DNA sequence demonstrated that other class-I genes were available as donors for interaction with the Kb gene to produce the mutations. Intriguingly, donor genes found in the K region (K1) and the D region (Db), as well as the Qa regions (Q4, Q10), were capable of the interactions. The amount of genetic transfer to Kb from other class-I donor genes may range from a potential minimum of 5 nucleotides to a potential maximum of 95 nucleotides. Genealogical analysis of several bm mutants has further indicated that at least some, if not all, of the gene interaction events generating Kb mutations occurred during mitotic amplification of the germ cells. Genetic recombination among class-I genes occurring in nature to the extent observed for the Kbm mutants could readily generate mosaic transplantation genes containing sequences derived from other class-I genes. Thus, it seems likely that genetic interaction plays a major role in the diversification and ongoing evolution of the MHC. The localization of altered amino acids in the in vivo mutant Kb molecules has directed our attention to recognition regions on the Kb product that play a major role in determining alloreactivity and H-2 associative recognition. The replacement of one or a few amino acids in either of the postulated recognition regions located in the alpha 1 domain (residues 70-90) or alpha 2 domain (residues 150-180) can have marked effects on biological function. While the majority of monoclonal antibodies recognize epitopes in one or the other recognition region, CTL recognize determinants dependent on the apparent interaction of amino acids located in both regions. These overall conclusions are supported to a large extent by studies on mutants derived from several sources, i.e. spontaneous mutants, mutagen-induced somatic variants, and products of hybrid H-2 genes. Studies of in vitro variants can provide a more refined approach for analysis of structure-function relationships through the introduction of minimal biochemical changes.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of yeast mutants with altered telomere structure.

Abstract: The chromosomes of the yeast Saccharomyces cerevisiae terminate in a tract of simple-sequence DNA [poly(C1-3A)] that is several hundred base pairs long. We describe the identification of mutant yeast strains that have telomeric tracts that are shorter than normal. A genetic analysis of these strains indicates that these short telomeres are the result of single nuclear recessive mutations and that these mutations can be classified into two different complementation groups. The full expression of the mutant phenotype shows a very long lag (approximately equal to 150 cell divisions). From our analysis of these mutants as well as other data, we suggest that the duplication of the telomeric poly(C1-3A) tract involves two processes, semiconservative replication and untemplated terminal addition of nucleotides.

Genetic evidence for a switching and energy-transducing complex in the flagellar motor of Salmonella typhimurium.

Abstract: The flaAII.2, flaQ, and flaN genes of Salmonella typhimurium are important for assembly, rotation, and counterclockwise-clockwise switching of the flagellar motor. Paralyzed and nonchemotactic mutants were subjected to selection pressure for partial acquisition of motility and chemotaxis, and the suppressor mutations of the resulting pseudorevertants were mapped and isolated. Many of the intergenic suppressor mutations were in one of the other two genes. Others were in genes for cytoplasmic components of the chemotaxis system, notably cheY and cheZ; one of the mutations was found in the cheA gene and one in a motility gene, motB. Suppression among the three fla genes was allele specific, and many of the pseudorevertants were either cold sensitive or heat sensitive. We conclude that the FlaAII.2, FlaQ, and FlaN proteins form a complex which determines the rotational sense, either counterclockwise or clockwise, of the motor and also participates in the conversion of proton energy into mechanical work of rotation. This switch complex is probably mounted to the base of the flagellar basal body and, via binding of the CheY and CheZ proteins, receives sensory information and uses it to control flagellar operation.

Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli.

Abstract: DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants. The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis. Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal-31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid. This indicates that mutations in the gyrB gene are responsible for nalidixic acid resistance.

A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen

Abstract: The nuclear gene for manganese-containing superoxide dismutase (MnSOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) of yeast mitochondria was mapped on chromosome VIII and inactivated by gene disruption. The resulting mutant lacked any protein cross-reacting with anti-MnSOD antibodies, and its mitochondria exhibited less than 1% of the cyanide-insensitive superoxide dismutase activity found in mitochondria of the wild-type parent strain. In the absence of oxygen, the mutant grew as rapidly as the wild-type parent. However, increasing concentrations of oxygen led to a progressive inhibition of growth. The properties of this mutant provide direct evidence that MnSOD contributes to the natural protection of cells against oxygen toxicity.

Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens.

Abstract: Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N-terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen.

Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives.

Abstract: We describe mutants of Escherichia coli that decrease the plasmid copy number of pBR322 derivatives. One mutant was partially characterized genetically and its mutation, designated pcnB for plasmid copy number, was mapped to approximately 3 min on the E. coli chromosome. This locus is distinct from other genes whose products are known to affect plasmid replication or stable plasmid maintenance. The pcnB mutant strain should be useful for cloning genes into pBR322 that have aberrant or deleterious effects on the cell when present in high copy number.

Infectious mutants of HTLV-III with changes in the 3' region and markedly reduced cytopathic effects

Abstract: A variant of human T-lymphotropic virus type III (HTLV-III) is described that replicates but does not kill normal human T cells in vitro. This variant, designated X10-1, was derived from the genome of a cytopathic HTLV-III clone (pHXB2D) by excision of a 200-base pair segment in the 3' region of the virus, spanning the env and 3'-orf genes. Comparable variants with 55 to 109 base pairs deleted exclusively in 3'-orf produced, in contrast, virus that was extremely cytopathic. On the basis of these findings it is concluded that the 3'-orf gene is not required for cytopathogenicity or replication of HTLV-III. In addition, the results suggest that virus replication and cytotoxicity are not intrinsically coupled. Furthermore, since clone X10-1 retains the ability to trans-activate genes linked to the viral long terminal repeats, trans-activation per se is not responsible for T-cell killing by HTLV-III. These results also raise the possibility that the carboxyl terminus of the envelope gene of HTLV-III has a direct role in T-cell killing by this virus.

Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

Abstract: The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.