Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase

Four aspartic acid residues (Asp126, Asp156, Asp233 and Asp295) of Bacillus cereus sphingomyelinase (SMase) in the conservative regions were changed to glycine by in vitro mutagenesis, and the mutant SMases [D126G (Asp126-->Gly etc.), D156G, D233G and D295G] were produced in Bacillus brevis 47, a protein-producing strain. The sphingomyelin (SM)-hydrolysing activity of D295G was completely abolished and those of D126G and D156G were reduced by more than 80%, whereas that of D233G was not so profoundly affected. Two mutant enzymes (D126G and D156G) were purified and characterized further. The hydrolytic activities of D126G and D156G toward four phosphocholine-containing substrates with different hydrophobicities, SM, 2-hexadecanoylamino-4-nitrophenylphosphocholine(HNP), lysophosphatidylcholine (lysoPC) and p-nitro-phenylphosphocholine (p-NPPC), were compared with those of the wild-type. The activity of D126G toward water-soluble p-NPPC was comparable with that of the wild-type. On the other hand, D156G catalysed the hydrolysis of hydrophilic substrates such as HNP and p-NPPC more efficiently (> 4-fold) than the wild-type. These results suggested that Asp126 and Asp156, located in the highly conserved region, may well be involved in a substrate recognition process rather than catalytic action. Haemolytic activities of the mutant enzymes were found to be parallel with their SM-hydrolysing activities. Two regions, including the C-terminal region containing Asp295, were found to show considerable sequence identity with the corresponding regions of bovine pancreatic DNase I. Structural predictions indicated structural similarity between SMase and DNase I. An evolutionary relationship based on the catalytic function was suggested between the structures of these two phosphodiesterases.

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Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase

Biochemical Journal ◽

10.1042/bj3010577 ◽

1994 ◽

Vol 301 (2) ◽

pp. 577-583 ◽

Cited By ~ 8

Author(s):

K Oda ◽

J Cheng ◽

T Saku ◽

N Takami ◽

M Sohda ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Endoplasmic Reticulum ◽

Aspartic Acid ◽

Chimeric Protein ◽

Attachment Site ◽

In Vitro Translation ◽

Placental Alkaline Phosphatase ◽

Wild Type ◽

Aspartic Acid Residue

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

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Effect of temperature on transition and transversion mutagenesis: characterization of wild type and a mutator T4 DNA polymerase mutant

Canadian Journal of Genetics and Cytology ◽

10.1139/g84-060 ◽

1984 ◽

Vol 26 (3) ◽

pp. 386-389 ◽

Cited By ~ 1

Author(s):

Linda J. Reha-Krantz ◽

Sükran Parmaksizoglu

Keyword(s):

Dna Polymerase ◽

Low Temperatures ◽

Bacteriophage T4 ◽

Effect Of Temperature ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Mutational Site

The effect of temperature on genetically well-defined mutational pathways was examined in the bacteriophage T4. The mutational site was a T4 rII ochre mutant which could revert to rII+ via a transversion or to the amber convertant via a transition. Temperature did not strongly affect any of the pathways examined in a wild-type background; however, increased temperature reduced the mutational activity of a mutator DNA polymerase mutant. Possible models to explain the role of temperature in mutagenesis are discussed as well as the significance of low temperatures for in vitro mutagenesis reactions.Key words: bacteriophage T4, mutator, transition, transversion, temperature effects.

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Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4087-4097.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4087-4097

Author(s):

J Wang ◽

N Suzuki ◽

Y Nishida ◽

T Kataoka

Keyword(s):

Heat Shock ◽

Adenylyl Cyclase ◽

Gene Replacement ◽

Yeast Cells ◽

Cyclase Activity ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Adenylyl Cyclase Activity

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

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Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes

Molecular and Cellular Biology ◽

10.1128/mcb.3.11.1996-2005.1983 ◽

1983 ◽

Vol 3 (11) ◽

pp. 1996-2005

Author(s):

R A Bhat ◽

B Metz ◽

B Thimmappaya

Keyword(s):

Dna Sequences ◽

Transcriptional Control ◽

Transcriptional Activity ◽

Evolutionary Relationship ◽

Trna Genes ◽

Wild Type ◽

Base Pairs ◽

Internal Promoter ◽

Cell Extracts

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.

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Specific protein binding to the simian virus 40 enhancer in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.2098 ◽

1986 ◽

Vol 6 (6) ◽

pp. 2098-2105 ◽

Cited By ~ 49

Author(s):

A G Wildeman ◽

M Zenke ◽

C Schatz ◽

M Wintzerith ◽

T Grundström ◽

...

Keyword(s):

Point Mutations ◽

Simian Virus 40 ◽

Simian Virus ◽

Specific Protein ◽

Dnase I ◽

Wild Type ◽

Dnase I Footprinting ◽

Nuclear Extracts

HeLa cell nuclear extracts and wild-type or mutated simian virus 40 enhancer DNA were used in DNase I footprinting experiments to study the interaction of putative trans-acting factors with the multiple enhancer motifs. We show that these nuclear extracts contain proteins that bind to these motifs. Because point mutations which are detrimental to the activity of a particular enhancer motif in vivo specifically prevent protection of that motif against DNase I digestion in vivo, we suggest that the bound proteins correspond to trans-acting factors involved in enhancement of transcription. Using mutants in which the two domains A and B of the simian virus 40 enhancer are either separated by insertion of DNA fragments or inverted with respect to their natural orientation, we also demonstrate that the trans-acting factors bind independently to the two domains.

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The Nonessential H2A N-Terminal Tail Can Function as an Essential Charge Patch on the H2A.Z Variant N-Terminal Tail

Molecular and Cellular Biology ◽

10.1128/mcb.23.8.2778-2789.2003 ◽

2003 ◽

Vol 23 (8) ◽

pp. 2778-2789 ◽

Cited By ~ 20

Author(s):

Qinghu Ren ◽

Martin A. Gorovsky

Keyword(s):

Tetrahymena Thermophila ◽

Gene Replacement ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Wild Type Protein ◽

Lysine Residues ◽

In The Wild ◽

Essential Function

ABSTRACT Tetrahymena thermophila cells contain three forms of H2A: major H2A.1 and H2A.2, which make up ∼80% of total H2A, and a conserved variant, H2A.Z. We showed previously that acetylation of H2A.Z was essential (Q. Ren and M. A. Gorovsky, Mol. Cell 7:1329-1335, 2001). Here we used in vitro mutagenesis of lysine residues, coupled with gene replacement, to identify the sites of acetylation of the N-terminal tail of the major H2A and to analyze its function in vivo. Tetrahymena cells survived with all five acetylatable lysines replaced by arginines plus a mutation that abolished acetylation of the N-terminal serine normally found in the wild-type protein. Thus, neither posttranslational nor cotranslational acetylation of major H2A is essential. Surprisingly, the nonacetylatable N-terminal tail of the major H2A was able to replace the essential function of the acetylation of the H2A.Z N-terminal tail. Tail-swapping experiments between H2A.1 and H2A.Z revealed that the nonessential acetylation of the major H2A N-terminal tail can be made to function as an essential charge patch in place of the H2A.Z N-terminal tail and that while the pattern of acetylation of an H2A N-terminal tail is determined by the tail sequence, the effects of acetylation on viability are determined by properties of the H2A core and not those of the N-terminal tail itself.

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Repression and activation of the Drosophila dopa decarboxylase gene in glia.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5659 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5659-5666 ◽

Cited By ~ 6

Author(s):

G S Mastick ◽

S B Scholnick

Keyword(s):

Germ Line ◽

Transcriptional Start Site ◽

Regulatory Region ◽

Regulatory Elements ◽

Dopa Decarboxylase ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Cell Activator ◽

Germ Line Transformation

Glial expression of the Drosophila dopa decarboxylase gene (Ddc) is repressed by a regulatory region located approximately 1 kb upstream of the transcriptional start site. We have used in vitro mutagenesis and germ line transformation to determine which elements within the Ddc promoter mediate repression. Our evidence suggests that the hypodermal cell activator elements IIA and IIB play a major role in the transcriptional regulation of Ddc in glial cells. A variety of mutations demonstrate that element IIA is a strong glial activator element and that element IIB is necessary for glial repression. Although these two regulatory elements are nearly identical in sequence, our data suggest that they are not redundant. Altering the wild-type number and spacing of elements IIA and IIB indicates that the wild-type arrangement of this repeat is critical for repression. We conclude that these key elements of the Ddc promoter regulate both activation and repression in glia.

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Tailless keratins assemble into regular intermediate filaments in vitro

Journal of Cell Science ◽

10.1242/jcs.97.2.317 ◽

1990 ◽

Vol 97 (2) ◽

pp. 317-324

Author(s):

M. Hatzfeld ◽

K. Weber

Keyword(s):

Intermediate Filaments ◽

Consensus Sequence ◽

In Vitro Mutagenesis ◽

Type I ◽

Type Ii ◽

Tail Domain ◽

Filament Formation ◽

Wild Type ◽

Keratin 8

To study the influence of the non alpha-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non alpha-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purify the protein by a two-step procedure. The filament-forming capacity of the truncated K8 with wild-type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occurring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs.

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Repression and activation of the Drosophila dopa decarboxylase gene in glia

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5659-5666.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5659-5666

Author(s):

G S Mastick ◽

S B Scholnick

Keyword(s):

Germ Line ◽

Transcriptional Start Site ◽

Regulatory Region ◽

Regulatory Elements ◽

Dopa Decarboxylase ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Cell Activator ◽

Germ Line Transformation

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In Vivo Analysis of the 3′ Untranslated Region of GB Virus B after In Vitro Mutagenesis of an Infectious cDNA Clone: Persistent Infection in a Transfected Tamarin

Journal of Virology ◽

10.1128/jvi.78.17.9389-9399.2004 ◽

2004 ◽

Vol 78 (17) ◽

pp. 9389-9399 ◽

Cited By ~ 29

Author(s):

Jae-Hwan Nam ◽

Kristina Faulk ◽

Ronald E. Engle ◽

Sugantha Govindarajan ◽

Marisa St. Claire ◽

...

Keyword(s):

Acute Hepatitis ◽

Persistent Infection ◽

Untranslated Region ◽

Acute Infection ◽

In Vitro Mutagenesis ◽

Short Sequence ◽

Wild Type ◽

Terminal Sequence

ABSTRACT GB virus B (GBV-B), the virus most closely related to hepatitis C virus (HCV), infects tamarins and causes acute hepatitis. The 3′ untranslated region (UTR) of an infectious GBV-B clone (pGBB) has a proximal short sequence followed by a poly(U) tract and a 3′ terminal sequence. Our investigators previously demonstrated that the 3′ terminal sequence was critical for in vivo infectivity. Here, we tested the effect of deleting the short sequence and/or the poly(U) tract from pGBB; infectivity of each mutant was tested by intrahepatic transfection of two tamarins with transcribed RNA. A mutant lacking both regions was not viable. However, mutants lacking either the short sequence or the poly(U) tract were viable. All four tamarins had a wild-type-like acute infection and developed acute hepatitis. Whereas we found that five tamarins transfected with the wild-type clone pGBB had acute resolving infection, one tamarin transfected with the poly(U) deletion mutant became persistently infected. This animal had viremia and hepatitis until its death at week 90. The genomes recovered at weeks 2, 7, 15, 20, 60, and 90 lacked the poly(U) stretch. Eight amino acid changes were identified at week 90. One change, in the putative p7 protein, was dominant at week 15. Thus, persistence of GBV-B, like persistence of HCV, was associated with the emergence of virus variants. Four tamarins inoculated with serum collected at weeks 2 and 90 from the tamarin with persistent infection had an acute resolving infection. Nonetheless, the demonstration that GBV-B can persist in tamarins strengthens its relevance as a surrogate model for the study of HCV.

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