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PubMed2000

Fliih, the murine homologue of the Drosophila melanogaster flightless I gene: nucleotide sequence, chromosomal mapping and overlap with Llglh.

The Drosophila melanogaster flightless I gene is involved in cellularization processes in early embryogenesis and in the structural organization of indirect flight muscle. The encoded protein contains a gelsolin-like actin binding domain and an N-terminal leucine-rich repeat protein-protein interaction domain. We have cloned Fliih, the corresponding chromosomal gene from the mouse, and determined its nucleotide sequence (15.6 kb). The predicted Fliih protein of 1271 amino acids is 95% identical to the human FLII protein. Like the human gene, Fliih has 29 introns, compared with 13 in C. elegans and 3 in D. melanogaster. Fluorescence in situ hybridization was used to map Fliih to Chromosome 11B. Fliih lies adjacent to Llglh, the mouse homologue of the D. melanogaster tumor suppressor gene lethal(2) giant larvae. The sequence of the genomic DNA in this area, combined with cDNA sequences, establishes that the 3' ends of the Fliih and Llglh transcripts overlap. The overlap region contains polyA signals for both genes and is conserved between human and mouse.

Actins↗
PubMed1991

Molecular structure of the Dr adhesin: nucleotide sequence and mapping of receptor-binding domain by use of fusion constructs.

The Dr hemagglutinin of uropathogenic Escherichia coli mediates adherence to the upper urinary tract. E. coli strains which express this adhesin bind to the Dr blood group antigen and mediate mannose-resistant hemagglutination (MRHA). Chloramphenicol inhibits MRHA produced by the Dr hemagglutinin and may act as an analog for the tissue receptor at the adhesin-binding site. The nucleotide sequence of the Dr hemagglutinin fimbrial subunit was determined and found to have significant homology with that of F1845, a fimbrial adhesin associated with diarrhea, and with the afimbrial adhesin AFA-I of uropathogenic E. coli. Chimeric adhesin determinants consisting of the Dr structural subunit and F1845 accessory genes or of the F1845 structural subunit and Dr accessory genes were constructed. The Dr and F1845 determinants were shown to have a close structural relationship, with functional differences concentrated in the fimbrial subunit. Oligonucleotide-directed site-specific mutagenesis was used to facilitate construction of a hybrid adhesin subunit gene containing the amino terminus of F1845 fused to the carboxy terminus of the Dr structural gene. The resulting construct confers chloramphenicol-resistant hemagglutination when introduced into an E. coli strain expressing the cloned Dr hemagglutinin. The chloramphenicol sensitivity or resistant phenotype of MRHA produced by this family of adhesins is determined solely by the fimbrial subunit gene. Domains responsible for the chloramphenicol sensitivity of Dr-mediated MRHA reside within the amino-terminal portion of the fimbrial subunit.

Adhesins, Escherichia coli↗
PubMed1982

[Detection and mapping of conserved nucleotidic sequences between the genomes of human papillomavirus 1 a and bovine papillomavirus 1 by electron microscope heteroduplex analysis (author's transl)].

Three regions of partial homology have been detected between the genomes of human papillomavirus 1 a (HPV-1 a) and bovine papillomavirus 1 (BPV-1) by electron microscope analysis of heteroduplex molecules. These regions contain about 25% of non-homologous bases and represent 13% of the genome length. They have been mapped on the viral DNAs. This allowed a reciprocal orientation of the physical maps of the two genomes. Two regions are located in the middle of the transforming fragment of BPV-1 DNA and the third, at at diametrically opposite position, falls into the region of the genome coding most probably for a viral structural protein.

Animals↗
PubMed1999

Mapping cyclic nucleotide-induced conformational changes in cyclicAMP receptor protein by a protein footprinting technique using different chemical proteases.

CyclicAMP receptor protein (CRP) regulates transcription of numerous genes in Escherichia coli. Both cAMP and cGMP bind CRP, but only cAMP induces conformational changes that dramatically increase the specific DNA binding activity of the protein. We have shown previously that our protein footprinting technique is sensitive enough to detect conformational changes in CRP by cAMP [Baichoo N, Heyduk T. 1997. Biochemistry 36:10830-10836]. In this work, conformational changes in CRP induced by cAMP and cGMP binding were mapped and quantitatively analyzed by protein footprinting using iron complexed to diethylenetriaminepentaacetic acid ([Fe-DTPA]2-), iron complexed to ethylenediaminediacetic acid ([Fe-EDDA]), iron complexed to desferrioxamine mesylate ([Fe-HDFO]+), and copper complexed to o-phenanthroline ([(OP)2Cu]+) as proteases. These chemical proteases differ in size, charge, and hydrophobicity. Binding of cAMP to CRP resulted in changes in susceptibility to cleavage by all four proteases. Cleavage by [Fe-EDDA] and [Fe-DTPA]2- of CRP-cAMP detected hypersensitivities in the DNA-binding F alpha-helix, the interdomain hinge, and the ends of the C alpha-helix, which is involved in intersubunit interactions. [Fe-EDDA] and [Fe-DTPA]2- also detected reductions in cleavage in the D and E alpha-helices, which are involved in DNA recognition. Cleavage by [Fe-HDFO]+ of CRP-cAMP detected hypersensitivities in beta-strand 8, the B alpha-helix, as well as in parts of the F and C alpha-helices. [Fe-HDFO]+ also detected protections from cleavage in beta-strands 4 to 5 and their intervening loop, beta-strand 7, which is part of the nucleotide binding pocket, as well as in the D and E alpha-helices. Cleavage by [(OP)2Cu]+ of CRP-cAMP detected hypersensitivities in beta-strands 9 and 11 as well as in the D and E alpha-helices. [(OP)2Cu]+ also detected protections in the C alpha-helix , the interdomain hinge, and beta-strands 2-7. Binding of cGMP to CRP resulted in changes in susceptibility to cleavage only by [(OP)2Cu]+, which detected minor protections in beta-strands 3-7, the interdomain hinge, and the C alpha-helix. These results show that binding of cAMP causes structural changes in CRP in the nucleotide binding domain, the interdomain hinge, the DNA binding domain, and regions involved in intersubunit interaction. Structural changes induced by binding of cGMP appear to be very minor and confined to the nucleotide binding domain, the interdomain hinge, and regions involved in intersubunit interaction. Use of different cleaving agents in protein footprinting seems to give a more detailed picture of structural changes than the use of a single protease alone.

Cyclic AMP↗
PubMed1990

Human U1-70K ribonucleoprotein antigen gene: organization, nucleotide sequence, and mapping to locus 19q13.3.

We have isolated and sequenced the gene encoding the human U1-70K snRNP protein. U1-70K is an RNA-binding protein that is a specific component of the U1 small nuclear ribonucleoprotein complex (snRNP) and constitutes the major anti-(U1) RNP autoimmune antigen. We have mapped the U1-70K gene to the distal portion of chromosome 19, at band q13.3. The gene is greater than 44 kb in size and consists of 11 exons. The general structure of the gene has been completely conserved during vertebrate evolution and accounts for the production of several different U1-70K mRNA species by alternative pre-mRNA splicing. Comparison of the predicted amino acid sequences of animal U1-70K proteins reveals a high degree of conservation, particularly in the region of the RNP consensus domain. Even more striking is the complete conservation of the nucleotide sequence of an alternative included/excluded exon containing an in-frame translational termination codon. This conservation also includes significant portions of the downstream intervening sequence. This extraordinary conservation at the nucleotide sequence level suggests that alternative splicing of this exon serves an important function, perhaps in regulating the production of functional U1-70K protein.

Amino Acid Sequence↗
PubMed1984

Physical mapping and complete nucleotide sequence of the denV gene of bacteriophage T4.

Phage T4 deletion mutants that are folate analog resistant (far) and contain deletions in the region of the T4 genome near denV have been isolated previously. We showed that one of these mutants (T4farP12) expressed normal denV gene activity, whereas another mutant (T4farP13) was defective in the denV gene. The rII-distal (right) physical endpoints of these deletions defined the limits of the interval in which the rII-proximal (left) endpoint of the denV gene should be located. The deletion endpoints were identified by restriction and Southern hybridization analyses of phage derivatives containing deoxycytidine instead of hydroxymethyldeoxycytidine in their DNAs. The results of these analyses localized the rII-proximal (left) end of the denV gene to a region between 62.4 and 64.3 kilobases on the T4 physical map. denV+ phage resulted from marker rescue with two of five denV- alleles tested, using plasmids containing a 1.8-kilobase fragment from this region or a 179-base-pair terminal fragment derived from it. Sequencing of the 179-base-pair fragment from wild-type DNA showed a 130-base-pair open reading frame with its termination codon at the rII-proximal end. Confirmation that this open reading frame is part of the denV coding sequence was obtained by identifying a TAG amber codon in the homologous DNA derived from a denV amber mutant strain. This mutant strain rescued the denV+ allele from plasmids containing the wild-type sequence. An adjacent overlapping restriction fragment was also cloned, permitting determination of the remaining denV gene sequence. Based on these results, the 3' end of the coding region of the denV locus was mapped to kilobase position 64.07 on the T4 physical map, and the 5' end was mapped to position 64.48.

Base Sequence↗
PubMed1996

Nucleotide sequence, insertional mutagenesis, and transcriptional mapping of a conserved region of the baculovirus Autographa californica nuclear polyhedrosis virus (map unit 64.8-66.9).

The nucleotide sequence of a 2773-bp region of the Autographa californica nuclear polyhedrosis virus HR3 variant (AcMNPV) (map unit 64.4-68.2) has been determined. This region lies between the previously analyzed p6.9 and p80 genes. Three open reading frames were contained within this region, potentially coding for proteins of 45, 40, and 12 kDa. The 5' ends of transcripts capable of coding for the 45- and 12-kDa proteins map to consensus baculovirus late transcription start sites (ATAAG), while the major 5' end of the transcript coding for the 40-kDa protein maps 16 nucleotides downstream from another late transcription start site sequence, GTAAG. Attempts to prepare recombinant viruses containing insertions within these three genes were negative, suggesting that they are all essential for virus replication in cell culture. The size and organization of transcripts expressed from this region of AcMNPV were very similar to those of the previously described homologous region of Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV). The three AcMNPV protein sequences (p45, p40, and p12) showed amino acid sequence homology with the proteins p48, p45, and p12 of OpMNPV, suggesting this region is highly conserved in baculoviruses.

Amino Acid Sequence↗
PubMed2001

Nucleotide sequence, transcription map, and mutation analysis of the 13q14 chromosomal region deleted in B-cell chronic lymphocytic leukemia.

Deletions of the 13q14 chromosome region are associated with B-cell chronic lymphocytic leukemia (B-CLL) and several other types of cancer, suggesting the presence of a tumor suppressor gene. In previous studies the minimal region of deletion (MDR) was mapped to a less than 300-kilobase (kb) interval bordered by the markers 173a12-82 and 138G4/1.3R. For the identification of the putative tumor suppressor gene, the entire MDR (approximately 347 kb) has been sequenced, and transcribed regions have been identified by exon trapping, EST-based full-length complementary DNA cloning, database homology searches, and computer-assisted gene prediction analyses. The MDR contains 2 pseudogenes and 3 transcribed genes: CAR, encoding a putative RING-finger containing protein; 1B4/Leu2, generating noncoding transcripts; and EST70/Leu1, probably representing another noncoding gene (longest open reading frame of 78 codons). These genes have been sequenced in 20 B-CLL cases with 13q14 hemizygous deletion, and no mutations were found. Moreover, no somatic variants were found in the entire MDR analyzed for nucleotide substitutions by a combination of direct sequencing and fluorescence-assisted mismatch analysis in 5 B-CLL cases displaying 13q14-monoallelic deletion. The nondeleted allele of the CAR and EST70/Leu1 genes was expressed in B-CLL specimens, including those with monoallelic loss, whereas no expression of 1B4/Leu2 was detectable in B-CLL, regardless of the 13q14 status. These results indicate that allelic loss and mutation of a gene within the MDR is an unlikely pathogenetic mechanism for B-CLL. However, haplo-insufficiency of one of the identified genes may contribute to tumorigenesis. (Blood. 2001;97:2098-2104)

Animals↗
PubMed1987

Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-[alpha-32P]adenosine diphosphate. Evidence for two nucleotide binding sites in the beta subunit.

Photolabeling of nucleotide binding sites in nucleotide-depleted mitochondrial F1 has been explored with 2-azido [alpha-32P]adenosine diphosphate (2-N3[alpha-32P] ADP). Control experiments carried out in the absence of photoirradiation in a Mg2+-supplemented medium indicated the presence of one high affinity binding site and five lower affinity binding sites per F1. Similar titration curves were obtained with [3H]ADP and the photoprobe 3'-arylazido-[3H]butyryl ADP [( 3H]NAP4-ADP). Photolabeling of nucleotide-depleted F1 with 2-N3[alpha-32P]ADP resulted in ATPase inactivation, half inactivation corresponding to 0.6-0.7 mol of photoprobe covalently bound per mol F1. Only the beta subunit was photolabeled, even under conditions of high loading with 2-N3[alpha-32P]ADP. The identification of the sequences labeled with the photoprobe was achieved by chemical cleavage with cyanogen bromide and enzymatic cleavage by trypsin. Under conditions of low loading with 2-N3[alpha-32P]ADP, resulting in photolabeling of only one vacant site in F1, covalently bound radioactivity was located in a peptide fragment of the beta subunit spanning Pro-320-Met-358 identical to the fragment photolabeled in native F1 (Garin, J., Boulay, F., Issartel, J.-P., Lunardi, J., and Vignais, P. V. (1986) Biochemistry 25, 4431-4437). With a heavier load of photoprobe, leading to nearly 4 mol of photoprobe covalently bound per mol F1, an additional region of the beta subunit was specifically labeled, corresponding to a sequence extending from Gly-72 to Arg-83. The isolated beta subunit also displayed two binding sites for 2-N3-[alpha-32P]ADP. When F1 was first photolabeled with a low concentration of NAP4-ADP, leading to the covalent binding of 1.5 mol of NAP4-ADP/mol F1, with the bound NAP4-ADP distributed equally between the alpha and beta subunits, a subsequent photoirradiation in the presence of 2-N3[alpha-32P]ADP resulted in covalent binding of the 2-N3[alpha-32P]ADP to both alpha and beta subunits. It is concluded that each beta subunit in mitochondrial F1 contains two nucleotide binding regions, one of which belongs to the beta subunit per se, and the other to a subsite shared with a subsite located on a juxtaposed alpha subunit. Depending on the experimental conditions, the subsite located on the alpha subunit is either accessible or masked. Unmasking of the subsite in the three alpha subunits of mitochondrial F1 appears to proceed by a concerted mechanism.

Adenosine Diphosphate↗
PubMed2006

Modifications in Thermus thermophilus 23 S ribosomal RNA are centered in regions of RNA-RNA contact.

Ribosomal RNA from all organisms contains post-transcriptionally modified nucleotides whose function is far from clear. To gain insight into the molecular interactions of modified nucleotides, we investigated the modification status of Thermus thermophilus 5 S and 23 S ribosomal RNA by mass spectrometry and chemical derivatization/primer extension. A total of eleven modified nucleotides was found in 23 S rRNA, of which eight were singly methylated nucleotides and three were pseudouridines. These modified nucleotides were mapped into the published three-dimensional ribosome structure. Seven of the modified nucleotides located to domain IV, and four modified nucleotides located to domain V of the 23 S rRNA. All posttranscriptionally modified nucleotides map in the center of the ribosome, and none of them are in contact with ribosomal proteins. All except one of the modified nucleotides were found in secondary structure elements of the 23 S ribosomal RNA that contact either 16 S ribosomal RNA or transfer RNA, with five of these nucleotides physically involved in intermolecular RNA-RNA bridges. These findings strongly suggest that the post-transcriptional modifications play a role in modulating intermolecular RNA-RNA contacts, which is the first suggestion on a specific function of endogenous ribosomal RNA modifications.

Binding Sites↗
PubMed1998

Ribozyme chemogenetics.

In this review I will outline several chemogenetic approaches used to determine the chemical basis of large ribozyme function and structure. The term chemogenetics was first used to describe site-specific functional group modification experiments in the analysis of DNA-protein interactions. Within the past few years equivalent experiments have been performed on large catalytic RNAs using both single-site substitution and interference mapping techniques with nucleotide analogues. While functional group mutagenesis is an important aspect of a chemogenetic approach, chemical correlates to genetic revertants and suppressors must also be realized for the genetic analogy to be intellectually valid and experimentally useful. Several examples of functional group revertants and suppressors have now been obtained within the Tetrahymena group I ribozyme. These experiments define an ensemble of tertiary hydrogen bonds that have made it possible to construct a detailed model of the ribozyme catalytic core. The model includes a functionally important monovalent metal ion binding site, a wobble-wobble receptor motif for helix-helix packing interactions, and a minor groove triple helix.

Models, Molecular↗
PubMed1991

A heptapeptide repeat contributes to the unusual length of chloroplast ribosomal protein S18. Nucleotide sequence and map position of the rpl33-rps18 gene cluster in maize.

The rpl33-rps18 gene cluster of the maize chloroplast genome has been mapped and sequenced. The derived amino acid sequence of the S18 protein shows a 7-fold repeat of a hydrophilic heptapeptide domain, S K Q P F R K, in the N-terminal region. Such a sequence is absent in the E. coli S18 and in the chloroplast S18 of the lower plant liverwort. In tobacco and rice chloroplast S18 it is present 2 and 6 times, respectively. Thus a long N-terminal repeat (resembling in composition the large C-terminal heptapeptide repeat in the eukaryotic pol II) appears to be characteristic of monocot cereal S18.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence, transcriptional mapping, and temporal expression of the gene encoding p39, a major structural protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata.

The gene encoding the 39-kDa major structural protein (p39) of Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) was sequenced and transcriptionally mapped, and its expression was examined at various times postinfection. By Northern hybridization, primer extension, and S1 nuclease analysis, we identified p39 mRNAs of approximately 2600 nt. By primer extension analysis, we identified two major sets of transcripts which initiated around -48 and -96 nt upstream of the translation start codon. The transcription start sites were located within the conserved baculovirus late gene consensus sequence, ATAAG, which is duplicated in the p39 5' flanking region. In OpMNPV-infected Lymantria dispar cells, the p39 mRNAs were expressed abundantly at 24 and 36 hr p.i. but were present in lower quantities at 48 hr p.i. The p39 gene contained an open reading frame of 1053 nt which encodes a predicted protein of 351 amino acids with an estimated molecular weight of 39.5 kDa. Three repeats of the amino acid sequence Ala-Pro-Ala-Ala-Pro were identified at the C-terminus of the predicted p39 protein.

Amino Acid Sequence↗
PubMed1993

Mapping the nucleotide-dependent conformational change of human N-ras p21 in solution by heteronuclear-edited proton-observed NMR methods.

Heteronuclear-edited proton-detected NMR methods are used to study the nucleotide-dependent conformational change between GDP- and GTP gamma S-bound forms of human N-ras p21. Amide groups of Asp are used as sensitive probes. When GTP gamma S is substituted for GDP in cellular N-ras p21, the chemical shifts of resonances Asp-47, -126, -154, and Asn-172, as well as Gly-77 and -151, are not sensitive to nucleotide exchange, whereas Asp-30, -33, -38, -54, -57, -69, -92, -105, and -119 are affected. Distinct chemical shift changes of Asp-33, -38, and -69 indicate that substantial structure changes occur in the effector-binding region and the switch II region. Crystallographic studies of H-ras p21 have indicated that the conformational differences are confined to residues 32-38 and 60-76. Our observations indicate that the nucleotide-dependent structural transitions of the protein in solution may not be identical to those in the crystal. They suggest that the peptide beyond Glu-76 participates in a conformational switch, and possibly is involved in effector function. We propose that the region roughly from Asp-92 to -105, and the region of guanine base-binding motif(s), e.g., 116NKXD, are candidate sites recognized by either a GDP/GTP release factor or a GTPase-affected protein.

Amino Acid Sequence↗