Nucleotide Segment Research Articles

Local and cis Effects of the H Element on Expression of Odorant Receptor Genes in Mouse

From the approximately 1,200 odorant receptor (OR) genes in the mouse genome, an olfactory sensory neuron is thought to express only one gene. The mechanisms of OR gene choice are not understood. A 2.1 kilobase region (the H element) adjacent to a cluster of seven OR genes has been proposed as a trans- and pan-enhancer for OR gene expression. Here, we deleted the H element by gene targeting in mice. The deletion abolishes expression of a family of three OR genes proximal to H, and H operates in cis on these genes. Deletion of H has a graded effect on expression of a distal group of four OR genes, commensurate with genomic distance. There is no demonstrable effect on expression of OR genes located outside the cluster. Our findings are not consistent with the hypothesis of H as an essential trans-acting enhancer for genome-wide regulation of OR gene expression.

Molecular epidemiology of rabies in Guangxi Province, south of China

Background Surveillance data for rabies in Guangxi Province in China showed that human rabies cases have gradually increased since 1996. Objective To evaluate the epidemiology of rabies at the molecular level and provide suggestions for effective prevention of rabies in Guangxi. Study design Since 2000, 1569 brains from suspected rabid animals were collected from different areas of Guangxi. Rabies virus was isolated from 42 samples. RT-PCR was used to amplify a 455 nucleotide segment of the 3′-terminal of the N gene. The sequencing data from that segment was used for phylogenetic analysis. Results Nucleotide homology comparisons and phylogenetic tree analysis based on this sequence indicated that all the rabies virus isolates from Guangxi belonged to genotype 1 and could be divided into four groups. Groups I, II and IV included 23, 10 and 8 isolates, respectively. These had nucleotide homologies of 97.1–100%, 98.2–100% and 99.1–99.6%, respectively. Only the GXN119 strain belonged to group III. Group I had two group-specific mutations: T90N and E110D. Group II had one group-specific mutation of T42S. Conclusions This study showed that rabies virus isolates from Guangxi have a close genetic relationship and topographical distribution.

Computational Analysis of RNA Editing Sites in Plant Mitochondrial Genomes Reveals Similar Information Content and a Sporadic Distribution of Editing Sites

A computational analysis of RNA editing sites was performed on protein-coding sequences of plant mitochondrial genomes from Arabidopsis thaliana, Beta vulgaris, Brassica napus, and Oryza sativa. The distribution of nucleotides around edited and unedited cytidines was compared in 41 nucleotide segments and included 1481 edited cytidines and 21,390 unedited cytidines in the 4 genomes. The distribution of nucleotides was examined in 1, 2, and 3 nucleotide windows by comparison of nucleotide frequency ratios and relative entropy. The relative entropy analyses indicate that information is encoded in the nucleotide sequences in the 5 prime flank (-18 to -14, -13 to -10, -6 to -4, -2/-1) and the immediate 3 prime flanking nucleotide (+1), and these regions may be important in editing site recognition. The relative entropy was large when 2 or 3 nucleotide windows were analyzed, suggesting that several contiguous nucleotides may be involved in editing site recognition. RNA editing sites were frequently preceded by 2 pyrimidines or AU and followed by a guanidine (HYCG) in the monocot and dicot mitochondrial genomes, and rarely preceded by 2 purines. Analysis of chloroplast editing sites from a dicot, Nicotiana tabacum, and a monocot, Zea mays, revealed a similar distribution of nucleotides around editing sites (HYCA). The similarity of this motif around editing sites in monocots and dicots in both mitochondria and chloroplasts suggests that a mechanistic basis for this motif exists that is common in these different organelle and phylogenetic systems. The preferred sequence distribution around RNA editing sites may have an important impact on the acquisition of editing sites in evolution because the immediate sequence context of a cytidine residue may render a cytidine editable or uneditable, and consequently determine whether a T to C mutation at a specific position may be corrected by RNA editing. The distribution of editing sites in many protein-coding sequences is shown to be non-random with editing sites clustered in groups separated by regions with no editing sites. The sporadic distribution of editing sites could result from a mechanism of editing site loss by gene conversion utilizing edited sequence information, possibly through an edited cDNA intermediate.

Simple & Rapid Generation of Complex DNA Profiles for the Undergraduate Laboratory

DNA profiles can be generated by a variety of techniques incorporating different types of DNA markers. Simple methods are commonly utilized in the undergraduate laboratory, but with certain drawbacks. Here, I present an advancement of the Alu dimorphism technique involving two tetraplex PCR analyses that yield 6,561 possible genotypes. This technique is simple to perform in an undergraduate laboratory. DNA markers are segments of nucleotides with known locations in the genome that can be informative in DNA profiling and genetic mapping studies if variations exist. Mini-satellites, a.k.a. variable number tandem repeats (VNTRs), consist of variants that are the result of 15-100 base pair (bp) repeated DNA fragments, and microsatellites are variants resulting from 2-7 bp repeated DNA fragments. VNTRs are commonly analyzed using the Southern blot procedure in order to detect numerous loci simultaneously, thereby generating a highly informative DNA profile. This is accomplished using a radioactive probe that consists of a short stretch of DNA nucleotides that are shared among the VNTR loci. Microsatellites require the use of high-resolution techniques such as standard acrylamide gel electrophoresis or the use of an automated DNA sequencer. Several microsatellite loci can be analyzed simultaneously by the multiplex polymerase chain reaction (PCR) technique. This methodology involves using primer pairs from several loci in a single reaction. Although there are commercial suppliers that offer kits for this technique, including the primers and markers for various alleles, cost becomes a limiting factor in undergraduate teaching laboratories. Based on cost, practicality, equipment availability, and isotope use, undergraduate laboratories are restricted in the types of analyses that can be performed. The two techniques typically used in the undergraduate laboratory are either the isolation of the VNTR locus referred to as D1S80, or an Alu dimorphism such as TPA-25 (Bloom et al., 1996), and are available as kits through commercial suppliers. Both simply involve PCR and analysis by electrophoresis on an agarose gel. D1S80 is a choice marker since it contains 29 different alleles yielding a possible 435 different genotypes [n (n+1)/2]. The primary drawback is the resolution limitation of agarose gels, creating difficulty in distinguishing individual alleles. Alu dimorphisms are variants that differ by the presence or absence of an Alu element. Alu elements are 300 bp retroposons (RNA-mediated transposable elements) that are amplified in the primate genome by the process of retrotransposition (retroposition) (Figure 1). The more recent integrations are not fixed in the human genome and therefore yield this type of variation (Roy-Engel et al., 2001), which has been useful in human population studies (Batzer et al., 1994; Roy-Engel et al., 2001) supporting the African origins of humans. The advantages of using the Alu dimorphism include its ease of use and simplicity of identification of the allelic variants. The drawback is the limited information attainable with only three possible genotypes (Figure 2). I previously developed a system to assay four Alu-containing loci using multiplex PCR (Kass, 2003). This is a simple, rapid technique for analyzing 81 ([3.sup.4]) possible genotypes. However, when using this for a simple forensic-type study in an undergraduate genomics laboratory, two individuals had the same genotype; and when incorporating this for a paternity test illustration, it was difficult to rule out one potential father. Therefore, I developed a second tetraplex reaction presented here for the first time. The two tetraplex reactions yield 6,561 combinations, dramatically increasing forensic and paternity capabilities of this tool. Additionally, the individual Alu variants that were chosen are referred to as intermediate frequency (IF) polymorphisms, i.e., both the presence and absence forms are commonly found among populations (Roy-Engel et al. …

Ab initio and DFT studies on nucleobase radicals of nucleotides

The nucleobase radicals are known for their role in producing nucleic acid strand break, and it is important to identify the radical formation at particular atomic site in the nucleobase centered radicals so that the major pathway for the nucleic acid damage may be trapped. Ab initio and DFT calculations of several nucleobase radicals formed at specific atomic site of nucleobases of a nucleotide segment have been performed. The radicals such as σ type and π type formed at different atomic sites of adenosine, guanosine, thymidine and cytidine occur at different energy levels, and also the π radicals are formed at lower energy level than the sigma radicals. Among the nucleobase radicals the C6 radical of thymidine is found to be the most stable as shown from the results of ROHF/6-31G** route. But the results obtained from DFT method do not show similar trend in energy values of ab initio calculations. Again the radical formation is monitored with respect to the location of radical center within Watson Crick (WC) and non Watson Crick (NWC) hydrogen bonding atomic sites of nucleobases. The nucleobase radicals formed at the atomic sites of non Watson Crick region are more stable than those of Watson Crick sites. The puckering of sugar and non-planarity of adenosine and guanosine nucleobases due to radical formation are found. The energies of forming the most stable radicals in nucleobases range from ∼−25 to −29 kcal/mol for ROHF/6-31G** route, whereas the energy values from DFT study range from −32 to −40 kcal/mol.

Rapid ATP-dependent Deadenylation of nanos mRNA in a Cell-free System from Drosophila Embryos

Shortening of the poly(A) tail (deadenylation) is the first and often rate-limiting step in the degradation pathway of most eukaryotic mRNAs and is also used as a means of translational repression, in particular in early embryonic development. The nanos mRNA is translationally repressed by the protein Smaug in Drosophila embryos. The RNA has a short poly(A) tail at steady state and decays gradually during the first 2-3 h of development. Smaug has recently also been implicated in mRNA deadenylation. To study the mechanism of sequence-dependent deadenylation, we have developed a cell-free system from Drosophila embryos that displays rapid deadenylation of nanos mRNA. The Smaug response elements contained in the nanos 3'-untranslated region are necessary and sufficient to induce deadenylation; thus, Smaug is likely to be involved. Unexpectedly, deadenylation requires the presence of an ATP regenerating system. The activity can be pelleted by ultracentrifugation, and both the Smaug protein and the CCR4.NOT complex, a known deadenylase, are enriched in the active fraction. The same extracts show pronounced translational repression mediated by the Smaug response elements. RNAs lacking a poly(A) tail are poorly translated in the extract; therefore, SRE-dependent deadenylation contributes to translational repression. However, repression is strong even with RNAs either bearing a poly(A) tract that cannot be removed or lacking poly(A) altogether; thus, an additional aspect of translational repression functions independently of deadenylation.

Matrix-Assisted Laser Desorption/Ionisation, Time-of-Flight Mass Spectrometry in Genomics Research

The beginning of this millennium has seen dramatic advances in genomic research. Milestones such as the complete sequencing of the human genome and of many other species were achieved and complemented by the systematic discovery of variation at the single nucleotide (SNP) and whole segment (copy number polymorphism) level. Currently most genomics research efforts are concentrated on the production of whole genome functional annotations, as well as on mapping the epigenome by identifying the methylation status of CpGs, mainly in CpG islands, in different tissues. These recent advances have a major impact on the way genetic research is conducted and have accelerated the discovery of genetic factors contributing to disease. Technology was the critical driving force behind genomics projects: both the combination of Sanger sequencing with high-throughput capillary electrophoresis and the rapid advances in microarray technologies were keys to success. MALDI-TOF MS–based genome analysis represents a relative newcomer in this field. Can it establish itself as a long-term contributor to genetics research, or is it only suitable for niche areas and for laboratories with a passion for mass spectrometry? In this review, we will highlight the potential of MALDI-TOF MS–based tools for resequencing and for epigenetics research applications, as well as for classical complex genetic studies, allele quantification, and quantitative gene expression analysis. We will also identify the current limitations of this approach and attempt to place it in the context of other genome analysis technologies.

DNA Repair: Dynamic Defenders against Cancer and Aging

You probably weren't thinking about your body's cellular DNA repair systems the last time you sat on the beach in the bright sunshine. Fortunately, however, while you were subjecting your DNA to the harmful effects of ultraviolet light, your cells were busy repairing the damage. The idea that our genetic material could be damaged by the sun was not appreciated in the early days of molecular biology. When Watson and Crick discovered the structure of DNA in 1953 [1], it was assumed that DNA is fundamentally stable since it carries the blueprint of life. However, over 50 years of research have revealed that our DNA is under constant assault by sunlight, oxygen, radiation, various chemicals, and even our own cellular processes. Cleverly, evolution has provided our cells with a diverse set of tools to repair the damage that Mother Nature causes. DNA repair processes restore the normal nucleotide sequence and DNA structure of the genome after damage [2]. These responses are highly varied and exquisitely regulated. DNA repair mechanisms are traditionally characterized by the type of damage repaired. A large variety of chemical modifications can alter normal DNA bases and either lead to mutations or block transcription if not repaired, more » and three distinct pathways exist to remove base damage. Base excision repair (BER) corrects DNA base alterations that do not distort the overall structure of the DNA helix such as bases damaged by oxidation resulting from normal cellular metabolism. While BER removes single damaged bases, nucleotide excision repair (NER) removes short segments of nucleotides (called oligonucleotides) containing damaged bases. NER responds to any alteration that distorts the DNA helix and is the mechanism responsible for repairing bulky base damage caused by carcinogenic chemicals such as benzo [a]pyrene (found in cigarette smoke and automobile exhaust) as well as covalent linkages between adjacent pyrimidine bases resulting from the ultraviolet (UV) component of sunlight. NER can be divided into two classes based on where the repair occurs. NER occurring in DNA that is not undergoing transcription (i.e., most of the genome) is called global genome repair (GGR or GGNER), while NER taking place in the transcribed strand of active genes is called transcription-coupled repair (TCR or TC-NER). We will explore NER in more detail below. Mismatch repair (MMR) is another type of excision repair that specifically removes mispaired bases resulting from replication errors. DNA damage can also result in breaks in the DNA backbone, in one or both strands. Single-strand breaks (SSBs) are efficiently repaired by a mechanism that shares common features with the later steps in BER. Double-strand breaks (DSBs) are especially devastating since by definition there is no intact complementary strand to serve as a template for repair, and even one unrepaired DSB can be lethal [3]. In cells that have replicated their DNA prior to cell division, the missing information can be supplied by the duplicate copy, or sister chromatid, and DSBs in these cells are faithfully repaired by homologous recombination involving the exchange of strands of DNA between the two copies. However, most cells in the body are non-dividing, and in these cells the major mechanism for repairing DSBs is by non-homologous end joining (NHEJ), which as the name implies involves joining two broken DNA ends together without a requirement for homologous sequence and which therefore has a high potential for loss of genetic information. « less

Modeling Oligonucleotide Microarray Signals

Chemical principles dictate that the specific binding of a target to its complementary probes on a DNA microarray surface, and the nonspecific binding between other nucleotide segments and the same probes, are mutually competitive. We demonstrate that this mechanism can be understood by considering the competitive chemical reaction taking place on the microarray surface. Inspired by the pioneering work of Zhang and Hekstra, we have developed a physical model for microarray signal analysis, based on possible reaction mechanisms, and implemented it with a parallel, generic, simulated-annealing algorithm. Using data supplied by the Affymetrix Latin-square spike-in experiments, our model showed excellent fitting of the data. This correlation between the predicted expression levels and the spike-in concentrations of test transcripts demonstrated good predictive abilities of our model.

Aldolases A and C Are Ribonucleolytic Components of a Neuronal Complex That Regulates the Stability of the Light-Neurofilament mRNA

A 68 nucleotide segment of the light neurofilament (NF-L) mRNA, spanning the translation termination signal, participates in regulating the stability of the transcript in vivo. Aldolases A and C, but not B, interact specifically with this segment of the transcript in vitro. Aldolases A and C are glycolytic enzymes expressed in neural cells, and their mRNA binding activity represents a novel function of these isozymes. This unsuspected new activity was first uncovered by Northwestern blotting of a brainstem/spinal cord cDNA library. It was confirmed by two-dimensional fractionation of mouse brain cytosol followed by Northwestern hybridization and protein sequencing. Both neuronal aldolases interact specifically with the NF-L but not the heavy neurofilament mRNA, and their binding to the transcript excludes the poly(A)-binding protein (PABP) from the complex. Constitutive ectopic expression of aldolases A and C accelerates the decay of a neurofilament transgene (NF-L) driven by a tetracycline inducible system. In contrast, mutant transgenes lacking mRNA sequence for aldolase binding are stabilized. Our findings strongly suggest that aldolases A and C are regulatory components of a light neurofilament mRNA complex that modulates the stability of NF-L mRNA. This modulation likely involves endonucleolytic cleavage and a competing interaction with the PABP. Interactions of aldolases A and C in NF-L expression may be linked to regulatory pathways that maintain the highly asymmetrical form and function of large neurons.

NF-κB mediates amyloid β peptide-stimulated activity of the human apolipoprotein E gene promoter in human astroglial cells

The apolipoprotein E gene (APOE) plays an important role in the pathogenesis of Alzheimer's disease (AD), and amyloid plaque comprised mostly of the amyloid-beta peptide (Aβ) is one of the major hallmarks of AD. However, the relationship between these two important molecules is poorly understood. We examined how Aβ treatment affects APOE expression in cultured cells and tested the role of the transcription factor NF-κB in APOE gene regulation. To delineate NF-κB's role, we have characterized a 1098 nucleotide (nt) segment containing the 5′-flanking region of the human APOE gene (−1054/+44, +1 transcription start site). Sequence analysis of this region suggests the presence of two potential NF-κB elements. To demonstrate promoter activity, the region was cloned upstream of a promoterless luciferase (reporter) gene. This segment was able to drive expression of luciferase in transient transfections of human fetal glial cells. Promoter activity was stimulated twofold by Aβ 1–40 (25 μM, 24 h) treatment. Pretreatment with double-stranded DNA decoy oligonucleotides against NF-κB (2 μM) reduced Aβ stimulation. Deletion and mutagenetic analyses demonstrated that the distal NF-κB element was functional and showed a strong DNA–protein complex band in gel shift analysis, similar to that from control NF-κB consensus element. An anti-inflammatory and anti-NF-κB drug, sodium salicylate, significantly blocked Aβ-induced APOE promoter function. Our data provide evidence that upregulation of APOE by Aβ in astroglial cells is mediated by an NF-κB-element present in the 5′-flanking region of the APOE gene.

Rapid differential diagnosis of Mycoplasma agalactiae and Mycoplasma bovis based on a multiplex-PCR and a PCR-RFLP

The membrane-protein 81 gene (mb-mp81) of Mycoplasma bovis was cloned, sequenced and compared to membrane-protein 81 gene (ma-mp81) of Mycoplasma agalactiae. After alignment of both sequences, specific primers pairs were designed from variable or unchanging nucleotide segments. In this study, we describe the development and optimization of a multiplex-PCR (MPCR) for the rapid detection of M. agalactiae and M. bovis strains. In addition, a simple and rapid PCR-restriction fragment length polymorphism (RFLP) assay, using the restriction enzymes AluI, DraI, RsaI and XbaI, is described to distinguish between both species. The results suggest that MPCR and PCR-RFLP assays could be used as an alternative method in routine diagnosis for rapid and specific simultaneous detection of M. agalactiae and M. bovis.

Smad1, β-catenin and Tcf4 associate in a molecular complex with the Myc promoter in dysplastic renal tissue and cooperate to control Myc transcription

Renal dysplasia, the major cause of childhood renal failure in humans, arises from perturbed renal morphogenesis and molecular signaling during embryogenesis. Recently, we discovered induction of molecular crosstalk between Smad1 and beta-catenin in the TgAlk3QD mouse model of renal medullary cystic dysplasia. Our finding that Myc, a Smad and beta-catenin transcriptional target and effector of renal epithelial dedifferentiation, is misexpressed in dedifferentiated epithelial tubules provided a basis for investigating coordinate transcriptional control by Smad1 and beta-catenin in disease. Here, we report enhanced interactions between a molecular complex consisting of Smad1, beta-catenin and Tcf4 and adjacent Tcf- and Smad-binding regions located within the Myc promoter in TgAlk3QD dysplastic renal tissue, and Bmp-dependent cooperative control of Myc transcription by Smad1, beta-catenin and Tcf4. Analysis of nuclear extracts derived from TgAlk3QD and wild-type renal tissue revealed increased levels of Smad1/beta-catenin molecular complexes, and de novo formation of chromatin-associated Tcf4/Smad1 molecular complexes in TgAlk3QD tissues. Analysis of a 476 nucleotide segment of the 1490 nucleotide Myc genomic region upstream of the transcription start site demonstrated interactions between Tcf4 and the Smad consensus binding region and associations of Smad1, beta-catenin and Tcf4 with oligo-duplexes that encode the adjacent Tcf- and Smad-binding elements only in TgAlk3QD tissues. In collecting duct cells that express luciferase under the control of the 1490 nucleotide Myc genomic region, Bmp2-dependent stimulation of Myc transcription was dependent on contributions by each of Tcf4, beta-catenin and Smad1. These results provide novel insights into mechanisms by which interacting signaling pathways control transcription during the genesis of renal dysplasia.

Molecular and pathological characterization of N:O isolates of the Potato virus Y from Manitoba, Canada

Previous studies have identified a recombinant isolate group of the Potato virus Y (PVY), PVYN:O, which exhibits a PVYO serotype and a PVYN pathotype. Molecular fingerprinting indicated that the isolates possess a European (Eu)–PVYN/NTN-like P1 gene and one PVYN-PVYO recombinant joint at the HC/Pro-P3 site. In this study, the complete genomic RNA sequence of two isolates of PVYN:O was determined. The overall identities at the polyprotein level between PVYN:O and other isolates ranged from 94.1% to 97.2%. However, the identities varied from 70.9% to 100% when individual mature proteins were compared. Scanning the nucleotide sequences revealed that PVYN:O comprises a recombinant genome in which the segment of nucleotides 1 to ~2400 is from Eu-PVYN, and the remainder from PVYO. Analysis of a population of PVYN:O isolates indicated the existence of pathological variants that induced different symptoms level in tobacco plants: severe, 16.7%; intermediate, 66.7%; and mild, 16.7%. Three representative isolates were selected for further analysis. Although pathologically different, the isolates were indistinguishable serotypically and genotypically when profiled using enzyme-linked immunosorbent assays and reverse transcription – polymerase chain reaction, respectively. No significant differences in nucleotide sequence were observed among the three isolates in 5'-UTR-P1-HC/Pro as well as the CP-3'-UTR region

Bone morphogenetic protein-2 suppresses collagenase-3 promoter activity in osteoblasts through a runt domain factor 2 binding site.

Transforming growth factor-beta (TGFbeta) superfamily of growth factors, which include bone morphogenetic proteins (BMPs), have multiple effects in osteoblasts. In this study, we examined the regulation of collagenase-3 promoter activity by BMP-2 in osteoblast-enriched (Ob) cells from fetal rat calvariae. BMP-2 suppressed the activity of a -2 kb collagenase-3 promoter/luciferase recombinant in a time- and dose-dependent manner. The BMP-2 effect on the collagenase-3 promoter was further tested in several collagenase-3 promoter deletion constructs and it was narrowed down to a -148 to -94 nucleotide segment of the promoter containing a runt domain factor 2 (Runx2) site at nucleotide -132 to -126. The effect of BMP-2 was obliterated in a collagenase-3 promoter/luciferase construct containing a mutated Runx2 (mRunx2) sequence indicating that the Runx2 site mediates the BMP-2 response. Electrophoretic mobility shift assays, using nuclear extracts from control and BMP-2-treated Ob cells, indicated that the Runx2 protein is a component of the specific DNA-protein complex formed on the Runx2 site and that the BMP-2 effect may be associated with minor protein modifications rather than major changes in the composition of specific proteins interacting with the Runx2 site. We confirmed that other members of the TGFbeta family can down-regulate the collagenase-3 promoter by showing that TGFbeta1 also suppresses the promoter activity in a time- and dose-dependent manner. In conclusion, this study demonstrates that BMP-2 and TGFbeta1 suppress collagenase-3 promoter activity in osteoblasts and establishes a link between BMP-2 action and collagenase-3 expression via Runx2, a major regulator of osteoblast formation and function.

Evidence for assembly-dependent folding of protein and RNA in an icosahedral virus

Ordered nucleic acid in an icosahedral virus was first visualized in the X-ray structure of the Picorna-like plant virus, Bean pod mottle virus (BPMV). Virus particles containing the 3500 nucleotide segment of the BPMV bipartite RNA genome (middle component) had nearly 20% of the genome ordered. Here we report the refined structures of the middle component, bottom component (particles containing the 5800 nucleotide segment of the genome), and top component (empty particles of BPMV capsid protein). The bottom component particles contain ordered RNA in the same location as middle component. Although the ordered RNA density in both nucleoprotein particles is the average of the contents of 60 icosahedral asymmetric units, both nucleoprotein components show that the base density for the first two nucleotides is predominantly purine, while the next five appear to be predominantly pyrimidine. The empty capsid demonstrates that RNA dictates the order of the N-terminal 19 residues of the large subunit because these residues are invisible in the top component.

A DNA sequence evolution analysis generalized by simulation and the markov chain monte carlo method implicates strand slippage in a majority of insertions and deletions.

To study the mechanisms for local evolutionary changes in DNA sequences involving slippage-type insertions and deletions, an alignment approach is explored that can consider the posterior probabilities of alignment models. Various patterns of insertion and deletion that can link the ancestor and descendant sequences are proposed and evaluated by simulation and compared by the Markov chain Monte Carlo (MCMC) method. Analyses of pseudogenes reveal that the introduction of the parameters that control the probability of slippage-type events markedly augments the probability of the observed sequence evolution, arguing that a cryptic involvement of slippage occurrences is manifested as insertions and deletions of short nucleotide segments. Strikingly, approximately 80% of insertions in human pseudogenes and approximately 50% of insertions in murids pseudogenes are likely to be caused by the slippage-mediated process, as represented by BC in ABCD --> ABCBCD. We suggest that, in both human and murids, even very short repetitive motifs, such as CAGCAG, CACACA, and CCCC, have approximately 10- to 15-fold susceptibility to insertions and deletions, compared to nonrepetitive sequences. Our protocol, namely, indel-MCMC, thus seems to be a reasonable approach for statistical analyses of the early phase of microsatellite evolution.

Identification of a new HLA-B allele, B*3705 containing a Bw6 sequence motif.

HLA-B37, which is Bw4 type antigen frequently found in linkage disequilibrium with A1, Cw6 and DR10 in all ethnic groups, generally has a very low frequency all over the world. We report a new HLA-B*37 allele, B*3705, identified in two potential bone marrow donors in the Korean population. B*3705, which has the Bw6 nucleotide segment, differs from B*3701 in three nucleotide positions: 311, 317 and 319 in exon2. The serological profile of B*3705 did not exhibit the B37 specificity. The putative haplotype associated with B*3705 in the Korean population could be A*02-Cw*0602-DRB1*1001-DQA1*0101-DQB1*0501-DPB1*02011.

Stem-loop SL4 of the HIV-1 Ψ RNA packaging signal exhibits weak affinity for the nucleocapsid protein. structural studies and implications for genome recognition

Encapsidation of the genome of the human immunodeficiency virus type-1 (HIV-1) during retrovirus assembly is mediated by interactions between the nucleocapsid (NC) domains of assembling Gag polyproteins and a ∼110 nucleotide segment of the genome known as the Ψ-site. The HIV-1 Ψ-site contains four stem-loops (SL1 through SL4), all of which are important for genome packaging. Recent isothermal titration calorimetry (ITC) studies have demonstrated that SL2 and SL3 are capable of binding NC with high affinity ( K d ∼ 140 nM), consistent with proposals for protein-interactive functions during packaging. To determine if SL4 may have a similar function, NC-interactive studies were conducted by NMR and gel-shift methods. In contrast to previous reports, we find that SL4 binds weakly to NC ( K d = (±14 μM), suggesting an alternative function. NMR studies indicate that the GAGA tetraloop of SL4 adopts a classical GNRA-type fold (R = purine, N = G, C, A or U), a motif that stabilizes RNA tertiary structures in other systems. In combination with previously reported gel mobility studies of Ψ-site deletion mutants, these findings suggest that SL4 functions in genome recognition not by binding to Gag, but by stabilizing the structure of the Ψ-site. Differences in the affinities of NC for SL2, SL3 and SL4 stem-loops can now be rationalized in terms of the different structural properties of stem loops that contain GGNG (SL2 and SL3) and GNRA (SL4) sequences.

The B 12-Binding Subunit of Glutamate Mutase from Clostridium tetanomorphum Traps the Nucleotide Moiety of Coenzyme B 12

Glutamate mutase from Clostridium tetanomorphum binds coenzyme B 12 in a base-off/His-on form, in which the nitrogenous ligand of the B 12-nucleotide function is displaced from cobalt by a conserved histidine. The effect of binding the B 12-nucleotide moiety to MutS, the B 12-binding subunit of glutamate mutase, was investigated using NMR spectroscopic methods. Binding of the B 12-nucleotide to MutS was determined to occur with K d=5.6(±0.7) mM and to be accompanied by a specific conformational change in the protein. The nucleotide binding cleft of the apo-protein, which is formed by a dynamic segment with propensity for partial α-helical conformation (the “nascent” α-helix), becomes completely structured upon binding of the B 12-nucleotide, with formation of helix α1. In contrast, the segment containing the conserved residues of the B 12-binding Asp-x-His-x-x-Gly motif remains highly dynamic in the protein/B 12-nucleotide complex. From relaxation studies, the time constant τ, which characterizes the time scale for the formation of helix α 1, was estimated to be about 30 μs 15N and was the same in both, apo-protein and nucleotide-bound protein. Thus, the binding of the B 12-nucleotide moiety does not significantly alter the kinetics of helix formation, but only shifts the equilibrium towards the structured fold. These results indicate MutS to be structured in such a way, as to be able to trap the nucleotide segment of the base-off form of coenzyme B 12 and provide, accordingly, the first structural clues as to how the process of B 12-binding occurs.