Polarity effects in the hisG gene of salmonella require a site within the coding sequence.

Abstract A single site in the middle of the coding sequence of the hisG gene of Salmonella is required for most of the polar effect of mutations in this gene. Nonsense and insertion mutations mapping upstream of this point in the hisG gene all have strong polar effects on expression of downstream genes in the operon; mutations mapping promotor distal to this site have little or no polar effect. Two previously known hisG mutations, mapping in the region of the polarity site, abolish the polarity effect of insertion mutations mapping upstream of this region. New polarity site mutations have been selected which have lost the polar effect of upstream nonsense mutations. All mutations abolishing the function of the site are small deletions; three are identical, 28-bp deletions which have arisen independently. A fourth mutation is a deletion of 16 base pairs internal to the larger deletion. Several point mutations within this 16-bp region have no effect on the function of the polarity site. We believe that a small number of polarity sites of this type are responsible for polarity in all genes. The site in the hisG gene is more easily detected than most because it appears to be the only such site in the hisG gene and because it maps in the center of the coding sequence.

Download Full-text

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1605336113 ◽

2016 ◽

Vol 113 (44) ◽

pp. 12508-12513 ◽

Cited By ~ 91

Author(s):

Bijoyita Roy ◽

Westley J. Friesen ◽

Yuki Tomizawa ◽

John D. Leszyk ◽

Jin Zhuo ◽

...

Keyword(s):

Premature Termination Codon ◽

Nonsense Suppression ◽

Base Pairs ◽

Inherited Disorders ◽

Nonsense Mutations ◽

Nonsense Codon ◽

Codon Positions ◽

A Site ◽

Cognate Trna ◽

Selection Of

A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren’s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren’s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

Download Full-text

The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

Biochemical Journal ◽

10.1042/bj2960851 ◽

1993 ◽

Vol 296 (3) ◽

pp. 851-857 ◽

Cited By ~ 23

Author(s):

T Belyaeva ◽

L Griffiths ◽

S Minchin ◽

J Cole ◽

S Busby

Keyword(s):

Escherichia Coli ◽

Point Mutations ◽

Growth Conditions ◽

Upstream Region ◽

E Coli ◽

Run Off ◽

A Site ◽

Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

Download Full-text

Bacteriophage P22 Antitermination boxB Sequence Requirements Are Complex and Overlap with Those of λ

Journal of Bacteriology ◽

10.1128/jb.00059-08 ◽

2008 ◽

Vol 190 (12) ◽

pp. 4263-4271 ◽

Cited By ~ 9

Author(s):

Alexis I. Cocozaki ◽

Ingrid R. Ghattas ◽

Colin A. Smith

Keyword(s):

Consensus Sequence ◽

Point Mutations ◽

Resonance Structure ◽

Moderate Activity ◽

Nuclear Magnetic Resonance Structure ◽

Bacteriophage P22 ◽

Base Pairs ◽

Rna Hairpins ◽

Transcription Antitermination ◽

Theories Of Evolution

ABSTRACT Transcription antitermination in phages λ and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the λ N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. A nuclear magnetic resonance structure of the P22 N peptide boxBleft complex and limited mutagenesis have been reported but do not reveal a consensus sequence for boxB. We have used a plasmid-based antitermination system to screen boxBs with random loops and to test boxB mutants. We find that P22 N requires boxB to have a GNRA-like loop with no simple requirements on the remaining sequences in the loop or stem. U:A or A:U base pairs are strongly preferred adjacent to the loop and appear to modulate N binding in cooperation with the loop and distal stem. A few GNRA-like hexaloops have moderate activity. Some boxB mutants bind P22 and λ N, indicating that the requirements imposed on boxB by P22 N overlap those imposed by λ N. Point mutations can dramatically alter boxB specificity between P22 and λ N. A boxB specific for P22 N can be mutated to λ N specificity by a series of single mutations via a bifunctional intermediate, as predicted by neutral theories of evolution.

Download Full-text

Mutations in the Fas Antigen in Patients With Multiple Myeloma

Blood ◽

10.1182/blood.v90.11.4266.4266_4266_4270 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4266-4270 ◽

Cited By ~ 8

Author(s):

Terry H. Landowski ◽

Ning Qu ◽

Ibrahim Buyuksal ◽

Jeffrey S. Painter ◽

William S. Dalton

Keyword(s):

Multiple Myeloma ◽

Fas Ligand ◽

Genetic Defect ◽

Point Mutations ◽

Neoplastic Disease ◽

Loss Of Function ◽

Fas Antigen ◽

Immune Effector ◽

Effector Cells ◽

A Site

Programmed cell death, or apoptosis, is well documented as a physiological means of eliminating activated lymphocytes and maintaining immune homeostasis. Apoptosis has also been implicated in the targeting of tumor cells by cytotoxic T lymphocytes and natural killer cells. One of the two primary mechanisms used in cell-mediated cytotoxicity is the Fas/FasLigand system. Activated or transformed cells expressing the Fas antigen on their surface are susceptible to killing by immune effector cells that express the Fas ligand. Many neoplastic cells, including those derived from patients with multiple myeloma, express Fas antigen on their surface, but do not undergo apoptosis in response to antigen crosslinking. One possibility for the lack of Fas-mediated apoptosis includes mutations in the Fas antigen. Loss of function mutations in the Fas antigen have been associated with congenital autoimmune disease in humans, and have been defined as the genetic defect the in lpr mice. Mutations in the Fas antigen have not been previously described in cancer patients. In this study, we show that mutations occur in the Fas antigen which may cause loss of function and contribute to the pathogenesis of the neoplastic disease, multiple myeloma. Using reverse transcriptase-polymerase chain reaction (RT-PCR), single-stranded conformation polymorphism (SSCP) analysis, and DNA sequencing, we examined the cDNA structure of the Fas antigen in 54 bone marrow (BM) specimens obtained from myeloma patients. Six patient specimens (11%) did not express detectable levels of Fas antigen mRNA. Of the 48 BM specimens which did express Fas antigen, 5 (10%) displayed point mutations. All of the mutations identified were located in the cytoplasmic region of the Fas antigen known to be involved in transduction of an apoptotic signal. Two separate individuals demonstrated an identical mutation at a site previously shown to be mutated in the congenital autoimmune syndrome, ALPS. One patient exhibited a point mutation at a site only two amino acids removed from the documented lesion of the lprcg mouse. Although the functional status of these point mutations remains to be determined, we propose that Fas antigen mutations may contribute to the pathogenesis and progression of myeloma in some patients.

Download Full-text

Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

Molecular and Cellular Biology ◽

10.1128/mcb.8.4.1398 ◽

1988 ◽

Vol 8 (4) ◽

pp. 1398-1407 ◽

Cited By ~ 70

Author(s):

M Guertin ◽

H LaRue ◽

D Bernier ◽

O Wrange ◽

M Chevrette ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Transient Expression ◽

Promoter Activity ◽

Gene Activation ◽

Point Mutations ◽

Binding Assays ◽

Recognition Sequence ◽

Base Pairs ◽

Receptor Recognition ◽

Afp Promoter

Mutations were introduced in 7 kilobases of 5'-flanking rat alpha 1-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.

Download Full-text

Mutations in the Fas Antigen in Patients With Multiple Myeloma

Blood ◽

10.1182/blood.v90.11.4266 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4266-4270 ◽

Cited By ~ 164

Author(s):

Terry H. Landowski ◽

Ning Qu ◽

Ibrahim Buyuksal ◽

Jeffrey S. Painter ◽

William S. Dalton

Keyword(s):

Multiple Myeloma ◽

Fas Ligand ◽

Genetic Defect ◽

Point Mutations ◽

Neoplastic Disease ◽

Loss Of Function ◽

Fas Antigen ◽

Immune Effector ◽

Effector Cells ◽

A Site

Abstract Programmed cell death, or apoptosis, is well documented as a physiological means of eliminating activated lymphocytes and maintaining immune homeostasis. Apoptosis has also been implicated in the targeting of tumor cells by cytotoxic T lymphocytes and natural killer cells. One of the two primary mechanisms used in cell-mediated cytotoxicity is the Fas/FasLigand system. Activated or transformed cells expressing the Fas antigen on their surface are susceptible to killing by immune effector cells that express the Fas ligand. Many neoplastic cells, including those derived from patients with multiple myeloma, express Fas antigen on their surface, but do not undergo apoptosis in response to antigen crosslinking. One possibility for the lack of Fas-mediated apoptosis includes mutations in the Fas antigen. Loss of function mutations in the Fas antigen have been associated with congenital autoimmune disease in humans, and have been defined as the genetic defect the in lpr mice. Mutations in the Fas antigen have not been previously described in cancer patients. In this study, we show that mutations occur in the Fas antigen which may cause loss of function and contribute to the pathogenesis of the neoplastic disease, multiple myeloma. Using reverse transcriptase-polymerase chain reaction (RT-PCR), single-stranded conformation polymorphism (SSCP) analysis, and DNA sequencing, we examined the cDNA structure of the Fas antigen in 54 bone marrow (BM) specimens obtained from myeloma patients. Six patient specimens (11%) did not express detectable levels of Fas antigen mRNA. Of the 48 BM specimens which did express Fas antigen, 5 (10%) displayed point mutations. All of the mutations identified were located in the cytoplasmic region of the Fas antigen known to be involved in transduction of an apoptotic signal. Two separate individuals demonstrated an identical mutation at a site previously shown to be mutated in the congenital autoimmune syndrome, ALPS. One patient exhibited a point mutation at a site only two amino acids removed from the documented lesion of the lprcg mouse. Although the functional status of these point mutations remains to be determined, we propose that Fas antigen mutations may contribute to the pathogenesis and progression of myeloma in some patients.

Download Full-text

Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.1.c189 ◽

1994 ◽

Vol 266 (1) ◽

pp. C189-C197 ◽

Cited By ~ 45

Author(s):

T. Ma ◽

H. Hasegawa ◽

W. R. Skach ◽

A. Frigeri ◽

A. S. Verkman

Keyword(s):

Base Pair ◽

Collecting Duct ◽

Rat Kidney ◽

Water Channel ◽

Cloning And Expression ◽

Base Pairs ◽

Kidney Medulla ◽

Coding Sequence ◽

Kidney Collecting Duct ◽

Protein Size

The cloning and expression of an apical membrane water channel from rat kidney collecting duct (WCH-CD) homologous to a 28-kDa integral membrane protein (CHIP28) was reported recently (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). We obtained an approximately 1.8-kilobase clone from a rat kidney lambda gt10 cDNA library by a polymerase chain reaction cloning method; whereas the coding sequence (814 base pairs, predicted protein size 29 kDa) was identical to that reported, we identified an in-frame ATG codon at base pair -123 predicting a protein size of 33 kDa. On Northern blots probed by cDNAs corresponding to the WCH-CD coding sequence (base pairs +1 to +814) or 5'-untranslated sequence (-403 to -16), a single band at 1.9 kilobases was observed in kidney medulla greater than in cortex but not in other tissues; mRNA expression was increased strongly by dehydration. Translation and oocyte expression studies were performed to identify the translation start site. The short (base pairs +1 to +814) and long (base pairs -123 to +814) cDNAs were subcloned in vector pSP64 containing the 5'-untranslated Xenopus globin sequence upstream to the ATGs; a 30-base pair c-myc sequence was engineered at the COOH- terminal for antibody recognition.(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text

Capture of a Transition State Using Molecular Dynamics: Creation of an Intercalation Site in dsDNA with Ethidium Cation

Journal of Nucleic Acids ◽

10.4061/2010/702317 ◽

2010 ◽

Vol 2010 ◽

pp. 1-4 ◽

Cited By ~ 7

Author(s):

Regina R. Monaco

Keyword(s):

External Surface ◽

Computational Study ◽

Helical Axis ◽

Base Pairs ◽

Bond Rotation ◽

Double Stranded Dna ◽

Dna Backbone ◽

Interaction Measure ◽

The Creation ◽

A Site

The mechanism of intercalation and the ability of double stranded DNA (dsDNA) to accommodate a variety of ligands in this manner has been well studied. Proposed mechanistic steps along this pathway for the classical intercalator ethidium have been discussed in the literature. Some previous studies indicate that the creation of an intercalation site may occur spontaneously, with the energy for this interaction arising either from solvent collisions or soliton propagation along the helical axis. A subsequent 1D diffusional search by the ligand along the helical axis of the DNA will allow the ligand entry to this intercalation site from its external, electrostatically stabilized position. Other mechanistic studies show that ethidium cation participates in the creation of the site, as a ligand interacting closely with the external surface of the DNA can cause unfavorable steric interactions depending on the ligands' orientation, which are relaxed during the creation of an intercalation site. Briefly, such a site is created by the lengthening of the DNA molecule via bond rotation between the sugars and phosphates along the DNA backbone, causing an unwinding of the dsDNA itself and separation between the adjacent base pairs local to the position of the ligand, which becomes the intercalation site. Previous experimental measurements of this interaction measure the enthalpic cost of this part of the mechanism to be about −8 kcal/mol. This paper reports the observation, during a computational study, of the spontaneous opening of an intercalation site in response to the presence of a single ethidium cation molecule in an externally bound configuration. The concerted motions between this ligand and the host, a dsDNA decamer, are clear. The dsDNA decamer AGGATGCCTG was studied; the central site was the intercalation site.

Download Full-text

Variability in nitrogen content of submerged aquatic vegetation: utility as an indicator of N dynamics within and among lakes

Water Science & Technology ◽

10.2166/wst.2012.065 ◽

2012 ◽

Vol 65 (7) ◽

pp. 1151-1157 ◽

Cited By ~ 1

Author(s):

Catherine Blanchet ◽

Gabriel Maltais-Landry ◽

Roxane Maranger

Keyword(s):

Nitrogen Content ◽

Plant Species ◽

Aquatic Ecosystems ◽

Submerged Aquatic Vegetation ◽

Aquatic Vegetation ◽

Spatial Scales ◽

Single Site ◽

N Availability ◽

N Dynamics ◽

A Site

Submerged aquatic vegetation (SAV) may serve as an integrative proxy of spatial and temporal nitrogen (N) availability in aquatic ecosystems as plants are physiologically capable of storing variable amounts of N. However, it is important to understand whether plant species behave similarly or differently within and among systems. We sampled different SAV species along a nutrient gradient at multiple sites within several lakes to determine variability in C:N ratios and % N content among species, among plants of the same species at a single site, among sites and among lakes. Species respond differently suggesting that not all plant types can be used universally as nutrient proxies. The greatest variability in % N and C:N ratios for Valliseneria americana was observed among lakes whereas for Elodea canadensis it was among sites within a lake and among plants within a site. This suggests that V. americana could be a particularly useful indicator of N availability at larger spatial scales (regional and within a large fluvial lake) but that E. canadensis was not a particularly useful proxy.

Download Full-text

Mutation of the p53 gene in human acute myelogenous leukemia

Blood ◽

10.1182/blood.v77.7.1500.1500 ◽

1991 ◽

Vol 77 (7) ◽

pp. 1500-1507 ◽

Cited By ~ 75

Author(s):

JM Slingerland ◽

MD Minden ◽

S Benchimol

Keyword(s):

Acute Myelogenous Leukemia ◽

P53 Protein ◽

Point Mutations ◽

P53 Gene ◽

Myelogenous Leukemia ◽

Wild Type ◽

P53 Expression ◽

Coding Sequence ◽

Acute Myelogenous ◽

Blast Cells

Abstract Heterogeneity of p53 protein expression is seen in blast cells of patients with acute myelogenous leukemia (AML). p53 protein is detected in the blasts of certain AML patients but not in others. We have identified p53 protein variants with abnormal mobility on gel electrophoresis and/or prolonged half-life (t 1/2). We have sequenced the p53 coding sequence from primary blast cells of five AML patients and from the AML cell line (OCIM2). In OCIM2, a point mutation in codon 274 was identified that changes a valine residue to aspartic acid. A wild type p53 allele was not detected in these cells. Two point mutations (codon 135, cysteine to serine; codon 246, methionine to valine) were identified in cDNA from blasts of one AML patient. Both mutations were present in blast colonies grown from single blast progenitor cells, indicating that individual leukemia cells had sustained mutation of both p53 alleles. The cDNAs sequenced from blast samples of four other patients, including one with prolonged p53 protein t 1/2 and one with no detectable p53 protein, were fully wild type. Thus, the heterogeneity of p53 expression cannot be explained in all cases by genetic change in the p53 coding sequence. The prolonged t 1/2 of p53 protein seen in some AML blasts may therefore reflect changes not inherent to p53. A model is proposed in which mutational inactivation of p53, although not required for the evolution of neoplasia, would confer a selective advantage, favoring clonal outgrowth during disease progression.

Download Full-text