Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes.

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

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Structural characterization of human aryl sulphotransferases

Biochemical Journal ◽

10.1042/bj3370337 ◽

1999 ◽

Vol 337 (2) ◽

pp. 337-343 ◽

Cited By ~ 18

Author(s):

Lulu A. BRIX ◽

Ronald G. DUGGLEBY ◽

Andrea GAEDIGK ◽

Michael E. McMANUS

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Change ◽

Substrate Binding ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Wild Type ◽

Substrate Specificities ◽

Loop Region ◽

Single Amino Acid Change

Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greater than 90% sequence identity, but vary markedly in their ability to catalyse the sulphonation of dopamine and p-nitrophenol. In order to investigate the amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes two regions of high sequence divergence, termed Regions A (amino acids 44–107) and B (amino acids 132–164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to change 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specificity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single amino acid change. However, compared with wild-type HAST1, a number of the mutations resulted in interference with substrate binding, as shown by elevated Ki values towards the co-substrate 3´-phosphoadenosine 5´-phosphosulphate, and in some cases loss of activity towards dopamine. These findings suggest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopamine, whereas a single amino acid at position 146 determines p-nitrophenol affinity. A HAST1 mutant was constructed to express a protein with four amino acids deleted (P87–P90). These amino acids were hypothesized to correspond to a loop region in close proximity to the substrate-binding pocket. Interestingly, the protein showed substrate specificities more similar to wild-type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.

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A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells.

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1751 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1751-1759 ◽

Cited By ~ 16

Author(s):

O R Choi ◽

C Trainor ◽

T Graf ◽

H Beug ◽

J D Engel

Keyword(s):

Amino Acid ◽

Precursor Cells ◽

Single Amino Acid ◽

Predicted Amino Acid Sequence ◽

Temperature Sensitive ◽

Wild Type ◽

Erythroid Precursor ◽

Single Amino Acid Change ◽

Avian Erythroblastosis Virus

A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.

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A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1751-1759.1986 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1751-1759

Author(s):

O R Choi ◽

C Trainor ◽

T Graf ◽

H Beug ◽

J D Engel

Keyword(s):

Amino Acid ◽

Precursor Cells ◽

Single Amino Acid ◽

Predicted Amino Acid Sequence ◽

Temperature Sensitive ◽

Wild Type ◽

Erythroid Precursor ◽

Single Amino Acid Change ◽

Avian Erythroblastosis Virus

A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.

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Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽

10.1139/g91-098 ◽

1991 ◽

Vol 34 (4) ◽

pp. 644-651 ◽

Cited By ~ 16

Author(s):

Kenneth Koo ◽

W. Dorsey Stuart

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Neurospora Crassa ◽

Rna Binding ◽

Signal Sequence ◽

Average Length ◽

Open Reading Frame ◽

Mrna Transcript ◽

S1 Nuclease ◽

Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

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Further characterization and determination of the single amino acid change in thetsI138reovirus thermosensitive mutant

Canadian Journal of Microbiology ◽

10.1139/w2012-033 ◽

2012 ◽

Vol 58 (5) ◽

pp. 589-595

Author(s):

Guy Lemay ◽

Martin Bisaillon

Keyword(s):

Amino Acid ◽

Amino Acid Change ◽

Genetic Alterations ◽

Biological Properties ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Nonpermissive Temperature ◽

Gene Encoding ◽

Viral Particles ◽

Single Amino Acid Change

Many temperature-sensitive mutants have been isolated in early studies of mammalian reovirus. However, the biological properties and nature of the genetic alterations remain incompletely explored for most of these mutants. The mutation harbored by the tsI138 mutant was already assigned to the L3 gene encoding the λ1 protein. In the present study, this mutant was further studied as a possible tool to establish the role of the putative λ1 enzymatic activities in viral multiplication. It was observed that synthesis of viral proteins is only marginally reduced, while it was difficult to recover viral particles at the nonpermissive temperature. A single nucleotide substitution resulting in an amino acid change was found; the position of this amino acid is consistent with a probable defect in assembly of the inner capsid at the nonpermissive temperature.

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Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3850-3859.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3850-3859

Author(s):

T A Coleman ◽

C Kunsch ◽

M Maher ◽

S M Ruben ◽

C A Rosen

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Fusion Protein ◽

Binding Specificity ◽

Single Amino Acid ◽

Nf Kappa B ◽

Dna Binding Specificity ◽

Dna Motif ◽

Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

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Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1711-1721.1986 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1711-1721

Author(s):

E M McIntosh ◽

R H Haynes

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequences ◽

Open Reading Frame ◽

Northern Analysis ◽

Hindiii Site ◽

Reading Frame ◽

Hindiii Restriction Site ◽

Cycle Dependent

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

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Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2001.281.4.g1034 ◽

2001 ◽

Vol 281 (4) ◽

pp. G1034-G1043 ◽

Cited By ~ 31

Author(s):

Kousei Ito ◽

Hiroshi Suzuki ◽

Yuichi Sugiyama

Keyword(s):

Amino Acids ◽

Multidrug Resistance ◽

Amino Acid ◽

Membrane Vesicles ◽

Single Amino Acid ◽

Sf9 Cells ◽

Transport Activity ◽

Wild Type ◽

Cationic Amino Acids ◽

The Difference

Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

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bimA encodes a member of the tetratricopeptide repeat family of proteins and is required for the completion of mitosis in Aspergillus nidulans

Journal of Cell Science ◽

10.1242/jcs.99.4.711 ◽

1991 ◽

Vol 99 (4) ◽

pp. 711-719

Author(s):

K.L. O'Donnell ◽

A.H. Osmani ◽

S.A. Osmani ◽

N.R. Morris

Keyword(s):

Amino Acid ◽

Aspergillus Nidulans ◽

Essential Gene ◽

Tetratricopeptide Repeat ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Amino Acid Residues ◽

Wild Type ◽

Reading Frame ◽

Integrative Transformation

The recessive, temperature-sensitive bimA1 mutation of Aspergillus nidulans blocks nuclei in metaphase at restrictive temperature. To determine whether the bimA product is essential, integrative transformation was used to create a mutation in the bimA gene. The mutation was maintained in a heterokaryon and the phenotype of spores produced by the heterokaryon was analyzed. Molecular disruption of the wild-type bimA gene is recessive in the heterokaryon and causes a metaphase block, demonstrating that bimA is an essential gene for mitosis. bimA was cloned by DNA-mediated complementation of its mutant phenotype at restrictive temperature, and the nucleotide sequence of a full-length cDNA was determined. A single large open reading frame was identified in the cDNA sequence, which predicts a protein containing 806 amino acid residues that is related (30.4% identity) to the Schizosaccharomyces pombe nuc2+ gene product, which also is required for completion of mitosis. The sequence of the bimA gene indicates that it is a member of a family of mostly nuclear proteins that contain a degenerate 34 amino acid repeat, the TPR (tetratricopeptide repeat) gene family.

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