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PubMed1988

Laotian (delta beta) (0)-thalassemia: molecular characterization of a novel deletion associated with increased production of fetal hemoglobin.

We have identified and molecularly characterized a novel deletion in the beta-globin gene cluster that increases fetal hemoglobin (HbF) synthesis in a 24-year-old Laotian man who is heterozygous for this mutation. The patient is asymptomatic with a mild anemia, hypochromia, and microcytosis (Ht = 39%, MCH = 22.8 pg, MCV = 71 fl), normal levels of HbA2 (3.0%) and 11.5% HbF (G gamma A gamma ratio 60 to 40), with heterocellular distribution (52% F cells). Extensive restriction endonuclease mapping defined the 5' breakpoint within the IVS II of the delta-globin gene, between positions 775 to 781 very similar to the 5' breakpoint of the Sicilian delta beta-thalassemia. However, the 3' breakpoint was localized between two Pst I sites 4.7 kb 3' of the beta-globin gene, thus ending about 0.7 kb upstream from the 3' breakpoint of the Sicilian delta beta-thalassemia. This results in a 12.5 kb deletion of DNA. It is of interest that the 5' breakpoint of the deletion residues within an AT-rich region which has been proposed as a specific recognition signal for recombination events, while the 3' breakpoint lies within a cluster of L1 repetitive sequences (formerly known as Kpn I family repeats). The presence of the 3' breakpoints of several other deletions within this region of L1 repeats also suggests that such sequences might serve as hot spots for recombination and eventually lead to thalassemia deletions. The similarity of the 5' and 3' breakpoints of these delta beta-thalassemias underscores the putative regulatory role of the deleted and juxtaposed sequences on the expression of the gamma-globin genes in adult life.

Adult↗
PubMed1989

Control of mucoidy in Pseudomonas aeruginosa: transcriptional regulation of algR and identification of the second regulatory gene, algQ.

A new alginate regulatory gene, algQ, was identified in a chromosomal region which, when tandemly amplified, induces mucoidy in Pseudomonas aeruginosa. The algQ gene was found closely linked to the previously identified algR gene. Both algQ and algR were required for transcription of the key alginate biosynthetic gene, algD. In addition, expression of the algR gene was studied. The algR promoter was mapped by S1 nuclease and reverse transcription and found to be activated in mucoid cells. However, even in nonmucoid cells, transcription of algR was detectable at an approximately 50-fold-lower level, as opposed to the algD promoter, which was silent in the nonmucoid background. Transcription of both promoters was studied by using algR- and algD-specific oligonucleotides and total cellular RNA from fresh cystic fibrosis isolates of mucoid P. aeruginosa and their nonmucoid revertants. Identical patterns of activity were found in all strains: in mucoid cells, both algR and algD were activated. This finding indicated that common mechanisms were involved in the regulation of alginate gene expression. However, when the algR gene was cloned behind the tac promoter on a broad-host-range-controlled expression vector, induction of transcription with isopropropyl-beta-D-thiogalactopyranoside (IPTG) caused the appearance of a nonmucoid phenotype in previously mucoid cells. This effect was transient, since removal of the inducer (IPTG) made cells mucoid again. Since the algR gene product is homologous to transcriptional regulators from a class of environmentally responsive systems (known to have a second, sensory component), the algQ gene could be a candidate for the sensory component of the alginate system.

Alginates↗
PubMed1989

Genetics and expression of the Q6 and Q8 genes. An LTR-like sequence in the 3' untranslated region.

A unique 2.2-kb mRNA is transcribed from the Q6 and Q8 genes of the mouse major histocompatibility complex. RNase protection experiments and DNA sequence analysis have mapped the 3' terminus to a site located 1110 bp downstream from exon 8. Comparison of the 3' sequence of Q8 to that of Q7 revealed that the two genes diverge from one another at a point located 200 bp into the untranslated domain. This finding explains the increased size of the transcript. RNase protection experiments involving twelve different strains of mice have revealed few sequence polymorphisms. A tissue distribution of the 2.2-kb transcript in B6 mice revealed that the highest quantities of message were present in the spleen with decreasingly lower amounts in the thymus, liver, kidney, testis, and brain. The H-2r haplotype appeared novel; it is phenotypically Qa-2-, yet expressed a 2.2-kb transcript which hybridizes to a Q8 probe. A comparison of a Qa-2hi expressor, B6, and a Qa-2low expressor, BALB/cFla, has established that these quantitative cell surface differences are reflected in mRNA differences. A homology search of the Genbank database has revealed that the 3' portion of Q8 contains extensive homology to a retrovirus-like long terminal repeat sequence that is characteristic of an embryonic-specific transposon (ETn).

Alleles↗
PubMed1990

DNA base changes in benzo[a]pyrene diol epoxide-induced dihydrofolate reductase mutants of Chinese hamster ovary cells.

We previously treated Chinese hamster ovary (CHO) cells with benzo[a]pyrene diol epoxide (BPDE) and mutants at the dihydrofolate reductase (dhfr) locus were isolated. On the basis of Southern blotting and RNA heteroduplex mapping experiments, 14 of the 15 mutants were presumed to carry point mutations. Two restriction fragment length polymorphism mutants were cloned and sequenced; one carried a point mutation, the other a -1 frameshift mutation. Using polymerase chain reaction techniques and direct sequencing of amplified mutant DNA, we have now determined the induced changes in the remaining 12 cell lines. All changes occurred at guanine bases; all target guanines except one were on the non-transcribed coding strand. Most mutants (79%) contained base substitutions; the rest (3/14) carried frameshift mutations. Of the point mutations, all but one (91%) were GC----TA transversions either in the dhfr coding sequence or at splice sites. The single exception was a GC----AT transition. Of the frameshift mutations, two were deletions of a GC pair and the other was an insertion of an AT pair. Four different mutations (29%) were clustered in a 3 bp region in exon 4. Tandem guanine bases adjacent to adenine were favored sites for mutation occurring in 9/14 cases (64%). These results are consistent with data previously obtained by others in the supF shuttle vector system and the CHO aprt gene.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1989

A new rearrangement of angiosperm chloroplast DNA in rye (Secale cereale) involving translocation and duplication of the ribosomal rpS15 gene.

The mapping and nucleotide sequencing of the rpS15 gene in the rye chloroplast DNA has shown that it is located in the inverted repeat (IR) and thus has two copies/genome. This is in contrast to tobacco and liverwort chloroplasts where rpS15 occurs as single-copy gene localized in the small single copy region (SSC). The direction of transcription of both gene copies in rye is toward SSC; that in tobacco and liverwort is toward IR-II. Further sequence data have revealed that the 3' end of each rye rpS15 gene copy is only 352 base pairs away from the corresponding IR.SSC junction and that this rearrangement event in rye involves also a 3' downstream-encoded and highly conserved chloroplast gene designated ORF393 in tobacco and ORF392 in liverwort. The latter in rye starts in both the IRs, but continues to full length into the SSC only from the IR-II. The direction of transcription of the nontruncated gene is fixed toward IR-I, being thus the inverse of ORF393/392 in tobacco and liverwort. Northern blot analysis has shown that the rearranged rpS15 gene is actively transcribed in rye chloroplasts and etioplasts and that its transcription pattern is different from that recently reported for tobacco rpS15.

Amino Acid Sequence↗
PubMed1987

A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping. I. Basic theory and an analysis of alcohol dehydrogenase activity in Drosophila.

Because some genes have been cloned that have a known biochemical or physiological function, genetic variation can be measured in a population at loci that may directly influence a phenotype of interest. With this measured genotype approach, specific alleles or haplotypes in the probed DNA region can be assigned phenotypic effects. In this paper we address several problems encountered in implementing the measured genotype approach with restriction site data. A number of analytical problems arise in part as a consequence of the linkage disequilibrium that is commonly encountered when dealing with small DNA regions: 1) different restriction site polymorphisms are not statistically independent, 2) the sites being measured are not likely to be the direct cause of the associated phenotypic effects, 3) haplotype classes may be phenotypically heterogeneous, and 4) the sites that are most strongly associated with phenotypic effects are not necessarily the most closely linked to the actual genetic cause of the effects. When recombination and gene conversion are rare, the primary cause of linkage disequilibrium is history (mutational origin, genetic drift, hitchhiking, etc.). We deal with historical association directly by producing a cladogram that partially reconstructs the evolutionary history of the present-day haplotype variability. The cladogram defines a nested analysis of variance that simultaneously detects phenotypic effects, localizes the effects within the cladogram, and identifies haplotypes that are potentially heterogeneous in their phenotypic associations. The power of this approach is illustrated by an analysis of the associations between alcohol dehydrogenase (ADH) activity and restriction site variability in a 13-kb fragment surrounding the ADH locus in Drosophila melanogaster.

Alcohol Dehydrogenase↗
PubMed1988

Spontaneous mitotic recombination in yeast: the hyper-recombinational rem1 mutations are alleles of the RAD3 gene.

The RAD3 gene of Saccharomyces cerevisiae is required for UV excision-repair and is essential for cell viability. We have identified the rem1 mutations (enhanced spontaneous mitotic recombination and mutation) of Saccharomyces cerevisiae as alleles of RAD3 by genetic mapping, complementation with the cloned wild-type gene, and DNA hybridization. The high levels of spontaneous mitotic gene conversion, crossing over, and mutation conferred upon cells by the rem1 mutations are distinct from the effects of all other alleles of RAD3. We present preliminary data on the localization of the rem1 mutations within the RAD3 gene. The interaction of the rem1 mutant alleles with a number of radiation-sensitive mutations is also different than the interactions reported for previously described (UV-sensitive) alleles of RAD3. Double mutants of rem1 and a defect in the recombination-repair pathway are inviable, while double mutants containing UV-sensitive alleles of RAD3 are viable. The data presented here demonstrate that: (1) rem1 strains containing additional mutations in other excision-repair genes do not exhibit elevated gene conversion; (2) triple mutants containing rem1 and mutations in both excision-repair and recombination-repair are viable; (3) such triple mutants containing rad52 have reduced levels of gene conversion but wild-type frequencies of crossing over. We have interpreted these observations in a model to explain the effects of rem1. Consistent with the predictions of the model, we find that the size of DNA from rem1 strains, as measured by neutral sucrose gradients, is smaller than wild type.

DNA Repair↗
PubMed1990

Molecular cloning and physical and functional characterization of the Salmonella typhimurium and Salmonella typhi galactose utilization operons.

The chromosomally encoded galactose utilization (gal) operons of Salmonella typhimurium and S. typhi were each cloned on similar 5.5-kilobase HindIII fragments into pBR322 and were identified by complementation of Gal- Escherichia coli strains. Restriction endonuclease analyses indicated that these Salmonellae operons share considerable homology, but some heterogeneities in restriction sites were observed. Subcloning and exonuclease mapping experiments showed that both operons have the same genetic organization as that established for the E. coli gal operon (i.e., 5' end, promoter, epimerase, transferase, kinase, and 3' end). Two gal operator regions (oE and oI) of S. typhimurium, identified by repressor titration in an E. coli superrepressor [galR(Sup)] mutant, were sequenced and found to flank the promoter region. This promoter region is identical to the -10 and -35 regions of the E. coli gal operon. Minicell studies demonstrated that the three gal structural genes of S. typhimurium encode separate polypeptides of 39 kilodaltons (kDa) (epimerase, 337 amino acids [aa's]), 41 kDa (transferase, 348 aa's), and 43 kDa (kinase, 380 aa's). Despite functional and organizational similarities, DNA sequence analysis revealed that the S. typhimurium gal genes show less than 70% homology to the E. coli gal operon. Because of codon degeneracy, the deduced amino acid sequences of these polypeptides are highly conserved (greater than 90% homology) as compared with those of the E. coli gal enzymes. These studies have defined basic genetic parameters of the gal genes of two medically important Salmonella species, and our findings support the hypothesized divergent evolution of E. coli and Salmonella spp. from a common ancestral parent bacterium.

Amino Acid Sequence↗
PubMed1989

Extreme heterogeneity in populations of vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) sequence evolution and population heterogeneity were examined by T1 oligonucleotide mapping. Individual clones isolated from clonal pools of wild-type Indiana serotype VSV displayed identical T1 maps. This was observed even after one passage at high concentrations of the potent viral mutagen 5-fluorouracil. Under low-multiplicity passage conditions, the consensus T1 fingerprint of this virus remained unchanged after 523 passages. Interestingly, however, individual clones from this population (passage 523) differed significantly from each other and from consensus sequence. When virus population equilibria were disrupted by high-multiplicity passage (in which defective interfering particle interference is maximized) or passage in the presence of mutagenic levels of 5-fluorouracil, rapid consensus sequence evolution occurred and extreme population heterogeneity was observed (with some members of these population differing from others at hundreds of genome positions). A limited sampling of clones at one stage during high-multiplicity passages suggested the presence of at least several distinct master sequences, the related subpopulations of which exhibit at least transient competitive fitness within the total virus population (M. Eigen and C.K. Biebricher, p. 211-245, in E. Domingo, J.J. Holland, P. Ahlquist, ed., RNA Genetics, vol. 3, 1988). These studies further demonstrate the important role of selective pressure in determining the genetic composition of RNA virus populations. This is true under equilibrium conditions in which little consensus sequence evolution is observed owing to stabilizing selection as well as under conditions in which selective pressure is driving rapid RNA virus genome evolution.

Animals↗
PubMed1987

Replication properties of pIM13, a naturally occurring plasmid found in Bacillus subtilis, and of its close relative pE5, a plasmid native to Staphylococcus aureus.

A naturally occurring plasmid from Bacillus subtilis, pIM13, codes for constitutively expressed macrolide-lincosamide-streptogramin B (MLS) resistance, is stably maintained at a high copy number, and exists as a series of covalent multimers. The complete sequence of pIM13 has been reported (M. Monod, C. Denoya, and D. Dubnau, J. Bacteriol. 167:138-147, 1986) and two long open reading frames have been identified, one of which (ermC') is greater than 90% homologous to the ermC MLS resistance determinant of the Staphylococcus aureus plasmid pE194. The second reading frame (repL) shares homology with the only long open reading frame of the cryptic S. aureus plasmid pSN2 and is probably involved in plasmid replication. The map of pIM13 is almost a precise match with that of pE5, a naturally occurring, stable, low-copy-number, inducible MLS resistance plasmid found in S. aureus. pIM13 is unstable in S. aureus but still multimerizes in that host, while pE5 is unstable in B. subtilis and does not form multimers in either host. The complete sequence of pE5 is presented, and comparison between pIM13 and pE5 revealed two stretches of sequence present in pE5 that were missing from pIM13. It is likely that a 107-base-pair segment in the ermC' leader region missing from pIM13 accounts for the constitutive nature of the pIM13 MLS resistance and that the lack of an additional 120-base-pair segment in pIM13 that is present on pE5 gives rise to the high copy number, stability, and multimerization in B. subtilis. The missing 120 base pairs occur at the carboxyl-terminal end of the putative replication protein coding sequence and results in truncation of that protein. It is suggested either that the missing segment contains a site involved in resolution of multimers into monomers or that the smaller replication protein causes defective termination of replication. It is concluded that pIM13 and pE5 are coancestral plasmids and it is probable that pIM13 arose from pE5.

Amino Acid Sequence↗
PubMed1987

Transcription of the alpha globin gene domain in normal and AEV-transformed chicken erythroblasts: mapping of giant globin-specific RNA including embryonic and adult genes.

The genomic domain of about 20 kbp of the chicken alpha-type globin genes, framed by AT-rich linkers (ATRLs; Moreau et al. 1982) and repetitive sequences (Broders et al. 1986), was cut into 13 fragments and subcloned. The in vitro labelled individual restriction fragments were used to test the extent of the transcribed domain by blot-hybridization of nuclear RNA in large excess from normal adult chicken and Avian Erythroblastosis Virus (AEV)-transformed erythroblasts. In both these types of cells, the AT-rich segments situated 6 kbp upstream of the first gene as well as all the domain including the embryonic pi and the adult alpha D and alpha A genes down to the AT-rich segment placed 3 kbp downstream were found to be transcribed. Electrophoresis of nuclear RNA, Northern blotting and hybridization with most of the nick-translated DNA probes revealed in all cases the presence of heterogeneous globin RNA molecules in the 3-12 kb range, as well as some distinct RNA bands. Single-stranded RNA probes of some genomic segments indicated asymmetrical transcription of the minus strand. A 12 kb globin-specific RNA including the pi and alpha A genes but not the intervening alpha D gene was observed in AEV-transformed cells: it includes sequences located far upstream and downstream from the alpha globin genes and might represent a processing product of a full length transcript spanning the whole domain. Reverse transcription by extension of primers placed in the first exon of each of the three globin genes confirmed the presence of continuous transcripts of the domain including the two adult and the embryonic globin genes.

Alpharetrovirus↗
PubMed2001

Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase.

A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated ras (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta-globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility.

Animals↗
PubMed1987

Relatively stable population of c-myc RNA that lacks long poly(A).

We examined the turnover of c-myc RNA in the human promyelocytic cell line HL-60. In whole-cell RNA from rapidly growing cells we observed two major size classes of c-myc RNA, 2.4 and 2.2 kilobases (kb). When HL-60 cells were treated with actinomycin D for 30 min to inhibit transcription, the 2.4-kb c-myc RNA population was rapidly degraded, while the 2.2-kb c-myc RNA was degraded much more slowly. S1 nuclease transcript mapping and promoter-specific probes were utilized to show that both the stable 2.2-kb and the labile 2.4-kb c-myc RNA populations have 5' ends at the second promoter site (P2) and 3' ends at the second poly(A) addition site. To examine further possible structural differences between these two RNA populations, we fractionated RNA on an oligo(dT)-cellulose column to separate RNAs that contained long poly(A) tails from those which did not. We found that the labile 2.4-kb c-myc RNA population bound to oligo(dT)-cellulose, while the more stable 2.2-kb c-myc RNA population did not. Preliminary estimates of their half-lives (t1/2) showed that the poly(A)+ c-myc RNA had a t1/2 of 12 min, while the c-myc RNA that did not bind to oligo(dT)-cellulose had a t1/2 of greater than 1 h. Several other cell types contain both poly(A)+ and nonpoly(A)+ c-myc RNAs including HeLa cells, normal human bone marrow cells, and normal mouse fetal liver cells. In agreement with the results in HL-60 cell, HeLa cell poly(A)+ c-myc RNA was more labile than c-myc RNA that lacked poly(A). The stable, nonpoly(A)+ c-myc RNA population may be important in the posttranscriptional regulation of c-myc expression.

Cell Line↗
PubMed1989

The size heterogeneity of rat insulin-like growth factor-I mRNAs is due primarily to differences in the length of 3'-untranslated sequence.

Previous studies have revealed multiple size classes of rat insulin-like growth factor-I (IGF-I) of estimated size 7.5-7.0, 1.9-1.5, and 1.2-0.9 kilobases (kb). Available sequence information accounts for only 2.1 kb of the 7.5-7.0 kb IGF-I mRNAs. We used oligomer directed ribonuclease H (RNase H) mapping to define the extent to which the unknown sequence in the large molecular weight mRNAs lies 5' or 3' to known sequence. Rat liver polyadenylated RNAs were incubated with oligomer probes complementary to internal rat IGF-I precursor (E domain) coding sequences. RNase H was used to hydrolyze IGF-I mRNAs at the point of annealment with the oligomers. Resultant 5' and 3'-IGF-I mRNA fragments were analyzed on Northern blots. A probe specific for type 1 (class C) 5'-sequences (the most predominant of multiple 5'-sequence types found on rat IGF-I mRNAs) identifies intact IGF-I mRNAs of 7.5-7.0, 1.9-1.5 and 1.2-0.9 kb but, after oligomer directed RNase cleavage of these mRNAs, identified only a single IGF-I mRNA 5'-fragment. Major differences in the length of sequence 5' to the IGF-I coding sequence therefore, do not account for the multiple size classes of type 1 (class C) IGF-I mRNAs. The size of the 5'-fragment suggests that the extent of sequence 5' to the IGF-I coding sequence is 0.4-0.7 kb in type 1 (class C) IGF-I mRNAs. Identification of multiple 3'-fragments of IGF-I mRNAs demonstrated heterogeneity in the 3'-ends of rat IGF-I mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Electron microscopy of nucleic acid.

Electron microscopy (EM) of nucleic acids was first described by Kleinschmidt and Zahn 1959 (1). By complexing duplex DNA with basic proteins and spreading the complexed DNA onto the surface of an aquous phase it was possible to obtain well stretched DNA molecules that could be examined by EM. Addition of formamide to DNA spreading solution as well as to hypophase prevented single-stranded DNA and RNA from forming secondary structures thus providing conditions for analysis of these molecules. EM studies have provided information about molecular weight, molecular configuration and homogeneity/heterogeneity of the DNA preparations examined. Furthermore, intramolecular heterogeneity in base sequence could be analyzed by partial denaturation of duplex DNA. By partial denaturation mapping information of the orientation of DNA molecules was obtained. Sequence homology between two DNA molecules were studied by heteroduplex analyses where the two DNAs to be studied were denatured to full strand separation followed by reassociation. This method has been widely used to analyze DNA mutations, and to discover sequence homologies within a DNA molecule or between two DNA molecules. Heteroduplex analyses between DNA and RNA (R-loop formation) provided information about distribution of ribosomal RNA genes and gave the first information of eukaryotic post transcriptional processing of mRNA. Also the various modes for DNA replication and transcription were studied by EM. It is possible to study duplex DNA not complexed by basic proteins by EM. The DNA is then adsorbed to an activated carbon film and rotary shadowed with finely grained metals to enhance the contrast. Using this method it was possible to study binding of proteins to DNA. This method was also used in the study of chromatin structure where it provided information of the size and distribution of nucleosomes as well as packaging of DNA in chromatin fibres. The complicated nature of DNA strand exchange and repair reactions was studied by in-vitro systems where EM analysis was used in the description of the presynoptic filament and structure of the joined molecules. Furthermore, EM provided the first direct evidence for the existence of the paranemic joint, where the DNA strands are joined without Watson-Crick base-pairing.

Animals↗
PubMed1987

Importance of conserved residues for the conformation of the T-loop in tRNAs.

The conformation of the T-loop of yeast tRNA(Asp) was studied by structural mapping techniques using chemical and enzymatic probes and by three-dimensional graphics modeling with the known crystallographic structures of tRNAs as references. The structural importance of C61 (conserved in the T-stem of all tRNAs) for the loop conformation was directly checked by ethylnitrosourea phosphate alkylation, either on the 3'-half tRNAAsp molecule or on a variant in which C61 was replaced by U61. The reactivity of P60 against ethylnitrosourea alkylation in the variant emphasizes the role of the hydrogen bond between this phosphate and position N4 of C61 for stabilizing the conformation of the T-loop. Experiments on several tRNA variants, containing C61 but altered in the sequence or in the length of the T-loop, indicate that other structural features help to stabilize the hydrogen bond network around P60. Evidence is presented that the reverse Hoogsteen base pair T54-A58 contributes to this stabilization by maintaining the hydrogen bonding between the 2'OH of ribose 58 and P60. Using graphics modeling and based on the chemical data. T-loops of several variants were constructed. It appears that both the constant length of the T-loop and the presence of psi 55 are crucial for the correct interaction between the T- and D-loops. The conclusion of this study is that the T-loop in tRNA possesses an intrinsic conformation (mainly governed by the constant residues) existing primarily without the structural context of the entire tRNA molecule.

Base Composition↗
PubMed2006

Mapping noncovalent ligand binding to stemloop domains of the HIV-1 packaging signal by tandem mass spectrometry.

The binding modes and structural determinants of the noncovalent complexes formed by aminoglycoside antibiotics with conserved domains of the HIV-1 packaging signal (Psi-RNA) were investigated using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The location of the aminoglycoside binding sites on the different stemloop structures was revealed by characteristic coverage gaps in the ion series obtained by sustained off-resonance irradiation collision induced dissociation (SORI-CID) of the antibiotic-RNA assemblies. The site positions were confirmed using mutants that eliminated salient structural features of the Psi-RNA domains. The effects of the mutations on the binding properties of the different substrates served to validate the position of the aminoglycoside site on the wild-type structures. Additional information was provided by docking experiments performed on the different aminoglycoside-stemloop complexes. The results have shown that, in the absence of features disrupting the regular A-helix of the double-stranded stem, aminoglycosides tend to bind in an area situated between the upper stem and the loop regions, as demonstrated for stemloop SL3. The presence of a tandem wobbles motif in SL4 modifies the regular geometry of the upper stem, which does not affect the general site location, but greatly increases its solution binding affinity compared with SL3. The platform motif in SL2 locates the binding site in the stem midsection and confers upon this stemloop an intermediate affinity toward aminoglycosides. In SL3 and SL4, the extensive overlap of the antibiotic site with the region used to bind the nucleocapsid (NC) protein provides the basis for a competition mechanism that could explain the aminoglycoside inhibition of the NC.SL3 and NC.SL4 assemblies. In contrast, the minimal overlap between the aminoglycoside and the NC sites in SL2 accounts for the absence of inhibition of the NC.SL2 complex.

Gas Chromatography-Mass Spectrometry↗
PubMed2002

Biophysical analysis of natural, double-helical DNA modified by a dinuclear platinum(II) organometallic compound in a cell-free medium.

Modifications of natural DNA by the dinuclear platinum(II) organometallic complex [[Pt(Me)Cl(Me(2)SO)](2)(mu-N-N)] [where N-N=H(2)N(CH(2))(6)NH(2)] (ORGANObisPt) were studied by methods of molecular biophysics. These methods include DNA binding studies using atomic absorption spectrophotometry, HPLC analysis of enzymatically digested DNA, interstrand cross-linking employing gel electrophoresis under denaturing conditions, DNA unwinding studied by gel electrophoresis, mapping of DNA adducts by transcription assay, DNA melting curves measured by absorption spectrophotometry and conformational analysis of platinated DNA by differential pulse polarography. The results indicate that the complex ORGANObisPt binds irreversibly to DNA. Its DNA binding mode is, however, different from that of the formally equivalent [[cis-PtCl(NH(3))(2)](2)(mu-N-N)] (1,1/c,c), which exhibits antitumor activity including that in the tumor cells resistant to cisplatin. Interestingly, ORGANObisPt binds to DNA considerably faster than 1,1/c,c and cisplatin. In addition, when ORGANObisPt binds to DNA it exhibits a strong base sequence specificity to guanine residues. ORGANObisPt forms mainly monofunctional adducts on double-helical DNA. It forms also a small amount of DNA interstrand cross-links (approximately 2%), i.e. a radically smaller amount in comparison with the complex 1,1/c,c. Importantly, these interstrand cross-links of ORGANObisPt are capable of terminating RNA synthesis in vitro, while its major monofunctional adducts are not. In addition, the adducts of ORGANObisPt affect the conformation of DNA, but in a different way than its dinuclear analogue 1,1/c,c or cisplatin. Some structural features of ORGANObisPt, such as the charge or nature of the trans and cis activating groups relative to the labile chloride, might be responsible for the altered DNA binding mode and biological activity in comparison with the 1,1/c,c compound.

Biophysical Phenomena↗