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PubMed2003

Shuffling of genes within low-copy repeats on 22q11 (LCR22) by Alu-mediated recombination events during evolution.

Low-copy repeats, or segmental duplications, are highly dynamic regions in the genome. The low-copy repeats on chromosome 22q11.2 (LCR22) are a complex mosaic of genes and pseudogenes formed by duplication processes; they mediate chromosome rearrangements associated with velo-cardio-facial syndrome/DiGeorge syndrome, der(22) syndrome, and cat-eye syndrome. The ability to trace the substrates and products of recombination events provides a unique opportunity to identify the mechanisms responsible for shaping LCR22s. We examined the genomic sequence of known LCR22 genes and their duplicated derivatives. We found Alu (SINE) elements at the breakpoints in the substrates and at the junctions in the truncated products of recombination for USP18, GGT, and GGTLA, consistent with Alu-mediated unequal crossing-over events. In addition, we were able to trace a likely interchromosomal Alu-mediated fusion between IGSF3 on 1p13.1 and GGT on 22q11.2. Breakpoints occurred inside Alu elements as well as in the 5' or 3' ends of them. A possible stimulus for the 5' or 3' terminal rearrangements may be the high sequence similarities between different Alu elements, combined with a potential recombinogenic role of retrotransposon target-site duplications flanking the Alu element, containing potentially kinkable DNA sites. Such sites may represent focal points for recombination. Thus, genome shuffling by Alu-mediated rearrangements has contributed to genome architecture during primate evolution.

Alu Elements↗

PubMed2005

A reaction landscape identifies the intermediates critical for self-assembly of virus capsids and other polyhedral structures.

The capsids of spherical viruses may contain from tens to hundreds of copies of the capsid protein(s). Despite their complexity, these particles assemble rapidly and with high fidelity. Subunit and capsid represent unique end states. However, the number of intermediate states in these reactions can be enormous-a situation analogous to the protein folding problem. Approaches to accurately model capsid assembly are still in their infancy. In this paper, we describe a sail-shaped reaction landscape, defined by the number of subunits in each species, the predicted prevalence of each species, and species stability. Prevalence can be calculated from the probability of synthesis of a given intermediate and correlates well with the appearance of intermediates in kinetics simulations. In these landscapes, we find that only those intermediates along the leading edge make a significant contribution to assembly. Although the total number of intermediates grows exponentially with capsid size, the number of leading-edge intermediates grows at a much slower rate. This result suggests that only a minute fraction of intermediates needs to be considered when describing capsid assembly.

Capsid Proteins↗

PubMed1995

Aerobic inactivation of fumarate reductase from Escherichia coli by mutation of the [3Fe-4S]-quinone binding domain.

Fumarate reductase from Escherichia coli functions both as an anaerobic fumarate reductase and as an aerobic succinate dehydrogenase. A site-directed mutation of E. coli fumarate reductase in which FrdB Pro-159 was replaced with a glutamine or histidine residue was constructed and overexpressed in a strain of E. coli lacking a functional copy of the fumarate reductase or succinate dehydrogenase complex. The consequences of these mutations on bacterial growth, assembly of the enzyme complex, and enzymatic activity were investigated. Both mutations were found to have no effect on anaerobic bacterial growth or on the ability of the enzyme to reduce fumarate compared with the wild-type enzyme. The FrdB Pro-159-to-histidine substitution was normal in its ability to oxidize succinate. In contrast, however, the FrdB Pro-159-to-Gln substitution was found to inhibit aerobic growth of E. coli under conditions requiring a functional succinate dehydrogenase, and furthermore, the aerobic activity of the enzyme was severely inhibited upon incubation in the presence of its substrate, succinate. This inactivation could be prevented by incubating the mutant enzyme complex in an anaerobic environment, separating the catalytic subunits of the fumarate reductase complex from their membrane anchors, or blocking the transfer of electrons from the enzyme to quinones. The results of these studies suggest that the succinate-induced inactivation occurs by the production of hydroxyl radicals generated by a Fenton-type reaction following introduction of this mutation into the [3Fe-4S] binding domain. Additional evidence shows that the substrate-induced inactivation requires quinones, which are the membrane-bound electron acceptors and donors for the succinate dehydrogenase and fumarate reductase activities. These data suggest that the [3Fe-4S] cluster is intimately associated with one of the quinone binding sites found n fumarate reductase and succinate dehydrogenase.

Aerobiosis↗

PubMed1971

Judgment of spatial orientation in patients with focal brain damage.

Thirty control and 121 brain-damaged patients with injury restricted to one hemisphere were presented with a test requiring the placing of a rod, fixed on a support by a hinged joint, in the same position as a model. Two versions of the test were given, one to be performed with the aid of vision and the other only by touch. The brain-damaged patients were subdivided into the following groups: right hemisphere patients without visual field defect (VFD): 30; right hemisphere patients with VFD: 20; left hemisphere patients without VFD: 46; left hemisphere patients with VFD: 25. The test modality did not differentially affect the performance of the groups. Both on the visually and on the tactually guided version of the test the right hemisphere group with VFD did significantly worse not only with respect to controls but also with respect to the three other braindamaged groups. The latter, in turn, were not significantly inferior to patients without cerebral damage. It appears, therefore, that when spatial perception is tested at a very basic and simple level, such as the detection of orientation in space of a rod, there is an almost complete dominance of the posterior region of the so-called minor hemisphere. This result must be contrasted with the less striking asymmetry of functions shown in more complex spatial tasks-for example, route finding, copying drawings, block designs-that are failed also by patients with damage to the left posterior area. Normal subjects reproduced the orientation of the rod on the horizontal plane with a constant error which was found to be dependent on the position of the model. Clockwise deviations were made when the model was on the left and counterclockwise deviations when the model was on the right. The constant error was present in left brain-damaged patients too, while it was not significantly different from zero in right brain-damaged patients. A systematic error was also found on the vertical plane and it consisted in the tendency to stop the rod below the level of the standard.

Brain Damage, Chronic↗

PubMed1997

The Bearded box, a novel 3' UTR sequence motif, mediates negative post-transcriptional regulation of Bearded and Enhancer of split Complex gene expression.

During the development of the Drosophila adult peripheral nervous system (PNS), inhibitory cell-cell interactions mediated by the Notch receptor are essential for proper specification of sensory organ cell fates. We have reported previously (M. W. Leviten, E. C. Lai and J. W. Posakony (1997) Development 124, 4039-4051) that the 3' untranslated regions (UTRs) of many genes involved in Notch signalling, including Bearded (Brd) and the genes of the Enhancer of split Complex (E(spl)-C), contain (often in multiple copies) two novel heptanucleotide sequence motifs, the Brd box (AGCTTTA) and the GY box (GTCTTCC). Moreover, the molecular lesion associated with a strong gain-of-function mutant of Brd suggested that the loss of these sequence elements from its 3' UTR might be responsible for the hyperactivity of the mutant gene. We show here that the wild-type Brd 3' UTR confers negative regulatory activity on heterologous transcripts in vivo and that this activity requires its three Brd box elements and, to a lesser extent, its GY box. We find that Brd box-mediated regulation decreases both transcript and protein levels, and our results suggest that deadenylation or inhibition of polyadenylation is a component of this regulation. Though Brd and the E(spl)-C genes are expressed in spatially restricted patterns in both embryos and imaginal discs, we find that the regulatory activity that functions through the Brd box is both temporally and spatially general. A Brd genomic DNA transgene with specific mutations in its Brd and GY boxes exhibits hypermorphic activity that results in characteristic defects in PNS development, demonstrating that Brd is normally regulated by these motifs. Finally, we show that Brd boxes and GY boxes in the E(spl)m4 gene are specifically conserved between two distantly related Drosophila species, strongly suggesting that E(spl)-C genes are regulated by these elements as well.

Animals↗

PubMed2004

Detection of extremely rare alleles by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (Bi-PAP-A): measurement of mutation load in mammalian tissues.

Pyrophosphorolysis-activated polymerization (PAP) was developed to detect extremely rare mutations in complex genomes. In theory, PAP can detect a copy of a single base mutation present in 3 x 10(11) copies of the wild-type allele. In practice, the selectivity of detection is limited by a bypass reaction involving a polymerase extension error from the unblocked oligonucleotide annealed to the opposing strand. Bidirectional PAP allele-specific amplification (Bi-PAP-A) is a novel method that uses two opposing 3'-terminal blocked pyrophosphorolysis-activatable oligonucleotides (P*s) with one nucleotide overlap at their 3' termini. This eliminates the problematic bypass reaction. The selectivity of Bi-PAP-A was examined using lambda phage DNA as a model system. Bi-PAP-A selectively detected two copies of a rare mutated allele in the presence of at least 2 x 10(9) copies of the wild-type lambda phage DNA. Bi-PAP-A was then applied to direct detection of spontaneous somatic mutations in the mouse genome at a frequency as low as 3 x 10(-9). A 370-fold variation in the frequency of a specific somatic mutation among different mouse samples was found, suggesting clonal expansion of mutation occurring during early development and a hyper-Poisson variance. Bi-PAP-A is a rapid, general, and automatable method for the detection of rare mutations.

Animals↗

PubMed2004

Tracking the recruitment of diabetogenic CD8+ T-cells to the pancreas in real time.

Development of autoimmune diabetes in both humans and mice is preceded by a prolonged period of inflammation of pancreatic islets by autoreactive T-cells. Noninvasive imaging techniques, including positron-emission tomography and optical or magnetic resonance imaging, have been used to track the recruitment of lymphocytes to sites of inflammation. These techniques, however, rely on labeling strategies that are non-antigen specific and do not allow specific tracking of the recruitment of autoreactive lymphocytes. Here we describe an antigen-specific magnetic label to selectively target a prevalent population of diabetogenic CD8(+) T-cells that contribute to the progression of insulitis to overt diabetes in NOD mice. Superparamagnetic nanoparticles coated with multiple copies of a high-avidity peptide/major histocompatibility complex ligand of these T-cells (NRP-V7/K(d)) are endocytosed by CD8(+) T-cells in an antigen-specific manner. Using these T-cells as probes, we show that inflammation of pancreatic islets by autoreactive T-cells can be detected in real time by magnetic resonance imaging. This study demonstrates the feasibility of visualizing the presence of ongoing autoimmune responses noninvasively.

Animals↗

PubMed1989

Adipokinetic hormone gene sequence from Manduca sexta.

A gene sequence encoding the neuropeptide adipokinetic hormone (AKH) was isolated from a genomic library of the tobacco hornworm, Manduca sexta. 2.5 x 10(5) recombinant plaques were screened with a 29-mer oligonucleotide substituted with deoxyinosine at six ambiguous codon positions. One clone was obtained from which we deduced an intronless gene encoding the AKH precursor (prepro-AKH). From NH2 to COOH terminus, prepro-AKH consists of a signal peptide, a single AKH peptide block followed by a Gly-Lys-Arg processing site, and a 34-residue sequence that appears unrelated to known peptides. The cloned AKH gene sequence hybridized to one transcript from the adult corpus cardiacum/corpus allatum complex. The Manduca genome appears to contain one copy of the AKH gene. These studies are a basis for explaining synthesis of a representative from a widespread, physiologically important family of arthropod neuropeptides.

Amino Acid Sequence↗

PubMed1995

Molecular cytogenetics of chronic myeloid leukemia with atypical t(6;9) (p23;q34) translocation.

We report the molecular cytogenetic analysis of a case of Philadelphia (Ph)-negative, BCR-positive chronic myeloid leukemia (CML) which appeared by conventional cytogenetics to have a t(6;9)(p23;q34) as the sole cytogenetic abnormality. Neither conventional nor pulse-field Southern blots detected any rearrangement of the DEK or CAN genes which are often fused in acute myeloid leukemia (AML) with t(6;9)(p23;q34). However, rearrangements of both BCR and ABL genes were detected. The breakpoint in BCR was located in the major translocation cluster region between exons b1 and b3. ABL rearrangements were detected with an ABL exon 1B probe and with a probe located 5' of the entire ABL gene. Comigration between the rearranged fragments obtained with M-bcr-5' and ABL exon 1B probes was observed, implying that the entire ABL gene was fused to the 5' part of the BCR gene. Fluorescence in situ hybridization (FISH) analyses using BCR and ABL probes showed that in 20% of metaphases BCR and ABL signals were present on one chromosome 6 at 6p23, whilst in 80% of metaphases BCR and ABL signals were identified on both copies of chromosome 6. Furthermore, FISH analysis with a whole-chromosome 22 paint demonstrated that chromosome 22 material was present on both copies of chromosome 6. These data indicate a complex Philadelphia translocation involving chromosome band 6p23 and duplication of the whole aberrant chromosome. The nature of the gene locus on 6p23, involved in this rearrangement, remains unknown. A similar translocation has been previously reported in a case of CML, which also lacked DEK and CAN gene rearrangements implying that abnormalities of 6p23 involving genes other than DEK may be a recurrent abnormality in CML.

Blotting, Southern↗

PubMed1995

[Neuropsychological comparison between dementia of Alzheimer type and multiple subcortical infarction by easily applicable test battery].

We compared the neuropsychological findings between dementia of Alzheimer type (DAT) and multiple subcortical infarction (MSI) using easily applicable test battery. Patients with DAT (N = 27) fulfilled the criteria of NINCDS-ADRDA for probable DAT and belonged to the mild degree in the DSM-III-R criteria for dementia. Patients with MSI (N = 30) scored more than 7 points in Hachinski Ischemic Score and were physically independent (Rankin Disability Scale < or = 3). Patients with DAT showed significantly more impaired performance than MSI in Mini-Mental State Examination, copy and delayed recall of Rey-Osterrieth Complex Figure Test and trial 5 of Rey Auditory-Verbal Learning Test, while patients with MSI achieved fewer categories and made more perseverative errors on Wisconsin Card Sorting Test than patients with DAT. Among these tests, delayed recall of Rey-Osterrieth Complex Figure Test seemed to be the most sensitive to discriminate DAT from MSI. These findings were thought to be compatible with the pathological facts that the degenerative process progresses from temporo-parietal lobe in DAT and frontal-subcortical region in MSI.

Aged↗

PubMed1996

The role of manganese in promoting multimerization and assembly of human immunodeficiency virus type 1 integrase as a catalytically active complex on immobilized long terminal repeat substrates.

The integration of a DNA copy of the viral genome into the genome of the host cell is an essential step in the replication of all retroviruses. Integration requires two discrete biochemical reactions; specific processing of each viral long terminal repeat terminus or donor substrate, and a DNA strand transfer step wherein the processed donor substrate is joined to a nonspecific target DNA. Both reactions are catalyzed by a virally encoded enzyme, integrase. A microtiter assay for the strand transfer activity of human immunodeficiency virus type 1 integrase which uses an immobilized oligonucleotide as the donor substrate was previously published (D. J. Hazuda, J. C. Hastings, A. L. Wolfe, and E. A. Emini, Nucleic Acids Res. 22;1121-1122, 1994). We now describe a series of modifications to the method which facilitate study of both the nature and the dynamics of the interaction between integrase and the donor DNA. The enzyme which binds to the immobilized donor is shown to be sufficient to catalyze strand transfer with target DNA substrates added subsequent to assembly; in the absence of the target substrate, the complex was retained on the donor in an enzymatically competent state. Assembly required high concentrations of divalent cation, with optimal activity achieved at 25 mM MnCl2. In contrast, preassembled complexes catalyzed strand transfer equally efficiently in either 1 or 25 mM MnCl2, indicating mechanistically distinct functions for the divalent cation in assembly and catalysis, respectively. Prior incubation of the enzyme in 25 mM MnCl2 was shown to promote the multimerization of integrase in the absence of a DNA substrate and alleviate the requirement for high concentrations of divalent cation during assembly. The superphysiological requirement for MnCl2 may, therefore, reflect an insufficiency for functional self-assembly in vitro. Subunits were observed to exchange during the assembly reaction, suggesting that multimerization can occur either before or coincident with but not after donor binding. These studies both validate and illustrate the utility of this novel methodology and suggest that the approach may be generally useful in characterizing other details of this biochemical reaction.

Base Sequence↗

PubMed1997

Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells.

Dendritic cells have the remarkable property of presenting any incoming antigen. To do so they must not only capture antigens with high efficiency and broad specificity, but must also maximize their capacity to load class II molecules of the major histocompatibility complex (MHC) with antigenic peptides in order to present a large array of epitopes from different proteins, each at a sufficient copy number. Here we show that formation of peptide-MHC class II complexes is boosted by inflammatory stimuli that induce maturation of dendritic cells. In immature dendritic cells, class II molecules are rapidly internalized and recycled, turning over with a half-life of about 10 hours. Inflammatory stimuli induce a rapid and transient boost of class II synthesis, while the half-life of class II molecules increases to over 100 hours. These coordinated changes result in the rapid accumulation of a large number of long-lived peptide-loaded MHC class II molecules capable of stimulating T cells even after several days. The capacity of dendritic cells to load many antigenic peptides over a short period of initial exposure to inflammatory stimuli could favour presentation of infectious antigens.

Antigen Presentation↗

PubMed2002

Ancestral polymorphism and adaptive evolution in the trichothecene mycotoxin gene cluster of phytopathogenic Fusarium.

Filamentous fungi within the Fusarium graminearum species complex (Fg complex) are the primary etiological agents of Fusarium head blight (scab) of wheat and barley. Scab is an economically devastating plant disease that greatly limits grain yield and quality. In addition, scabby grain is often contaminated with trichothecene mycotoxins that act as virulence factors on some hosts, and pose a serious threat to animal health and food safety. Strain-specific differences in trichothecene metabolite profiles (chemotypes) are not well correlated with the Fg complex phylogeny based on genealogical concordance at six single-copy nuclear genes. To examine the basis for this discord between species and toxin evolution, a 19-kb region of the trichothecene gene cluster was sequenced in 39 strains chosen to represent the global genetic diversity of species in the Fg complex and four related species of Fusarium. Phylogenetic analyses demonstrated that polymorphism within these virulence-associated genes is transspecific and appears to have been maintained by balancing selection acting on chemotype differences that originated in the ancestor of this important group of plant pathogens. Chemotype-specific differences in selective constraint and evidence of adaptive evolution within trichothecene genes are also reported.

DNA, Fungal↗

PubMed1989

Primate evolution of a human chromosome 1 hypervariable repetitive element.

The clone designated hMF #1 represents a clustered DNA family, located on chromosome 1, consisting of tandem arrays displaying a monomeric length of 40 bp and a repetition frequency of approximately 7 x 10(3) copies per haploid genome. The sequence hMF #1 reveals multiple restriction fragment length polymorphisms (RFLPs) when human genomic DNA is digested with a variety of 4-6-bp recognition sequence restriction enzymes (i.e., Taq I, Eco RI, Pst I, etc.). When hamster and mouse genomic DNA was digested and analyzed, no cross-species homology could be observed. Further investigation revealed considerable hybridization in the higher primates (chimpanzee, gorilla, and orangutan) as well as some monkey species. The evolutionary relationship of this repetitive DNA sequence, found in humans, to that of other primates was explored using two hybridization methods: DNA dot blot to establish copy number and Southern DNA analysis to examine the complexity of the RFLPs. Homology to the hMF #1 sequence was found throughout the suborder Anthropoidea in 14 ape and New and Old World monkey species. However the sequence was absent in one species of the suborder Prosimii. Several discrepancies between "established" evolutionary relationships and those predicted by hMF #1 exist, which suggests that repetitive elements of this type are not reliable indicators of phylogenetic branching patterns. The phenomenon of marked diversity between sequence homologies and copy numbers of dispersed repetitive DNA of closely related species has been observed in Drosophila, mice, Galago, and higher primates. We report here a similar phenomenon for a clustered repeat that may have originated at an early stage of primate evolution.

Animals↗

PubMed1998

A maize Myb homolog is encoded by a multicopy gene complex.

The maize P gene encodes a Myb-homologous transcriptional regulator of flavonoid pigmentation in floral organs, and different P gene alleles condition precise tissue- and organ-specific pigmentation patterns. To determine the molecular basis for allele-specific expression patterns, we have isolated and compared two natural alleles of the P gene which differ in expression, structure and copy number. The P-rr allele is associated with pigmentation of most floral tissues and contains a single copy of the P gene. In contrast, the P-wr allele restricts pigmentation to a subset of floral tissues, and is composed of six gene copies arranged in a tandem head-to-tail array. Each of the six repeats contains a single P gene, including regulatory and coding sequences. Despite the six-fold tandem repetition of P-wr gene copies, P-wr mRNA levels in kernel pericarp are much reduced compared to mRNA levels from the single-copy P-rr gene. Moreover, the P-wr multicopy complex is hypermethylated relative to P-rr. Thus, maize P gene alleles may represent a natural system for studying the effects of methylation and gene copy number on tissue-specific gene expression. We discuss the possibility that somatic pairing of repeated gene copies may be involved in regulating gene expression.

Alleles↗

PubMed1976

An enzyme complex for the dehydrogenation of phytoene in Phycomyces.

Phycomyces strain C5, carrying mutation carB10, accumulates phytoene instead of beta-carotene. Heterokaryons containing C5 nuclei and different other nuclei carrying the wild type carB allele accumulate significant amounts of phytofluene, zota-carotene and neurosporene. From quantitative analyses of carotenes and nuclear proportions in the heterokaryons we conclude that four copies of the carB gene product, assembled in an enzyme complex, act sequentially in the conversion of phytoene to lycopene.

Carotenoids↗

PubMed1990

Immune deficiency due to high copy numbers of an Ak beta transgene.

Because allelic polymorphism of the major histocompatibility complex class II antigens affects the immune response at several levels, we wished to characterize the contribution of a particular alpha or beta chain in vivo using transgenic mice. We have established and characterized 12 lines of H-2s/s mice carrying from 1 to 65 copies of an Ak beta transgene. The transgene was coexpressed with the endogenous allele in a tissue-specific manner, and Ak beta mRNA expression correlated well with transgene copy number. High copy number (extreme overexpression) of the transgene was associated with a variety of defects, including a significant reduction in Ia cell-surface expression, a severe decrease in B-cell number, abnormal extramedullary granulopoiesis, and an increased susceptibility to infection. In this paper we describe in detail the phenotype associated with high copy numbers of the Ak beta transgene. The defects we have observed may be relevant to similar phenomena seen in other transgenic mice. In addition, these mice have fortuitously provided a system in which to assess the effect of various levels of class II cell-surface expression in the thymus on selection of the T-cell repertoire.

Alleles↗

PubMed1992

Definition and functional implications of gibberellin and abscisic acid cis-acting hormone response complexes.

The mechanisms by which cis-acting hormone response elements affect transcription is unclear. In this study, we demonstrated that a second "coupling element," identified as O2S, must be present to allow a single copy of either the gibberellin response element (GARE) or the abscisic acid response element (ABRE) to mediate their hormonal effects in the barley Amy32b alpha-amylase gene promoter. The interactive effects of the O2S and the GARE are constrained positionally and spatially; thus, together they form a gibberellin response complex (GARC). The absolute requirement of the O2S for function of the ABRE demonstrates that these together form an abscisic acid response complex (ABRC). A second copy of the GARE can substitute for the O2S in the GARC, but only in one orientation. By expressing the GARC-containing and ABRC-containing promoters in developing aleurone tissue, we showed that hormonal effects prevent alpha-amylase gene expression during the second half of grain development, but other mechanisms suppress expression earlier. Our results suggest that the specific sequence serving as a coupling element in a given gene promoter will greatly affect where and when the GARE or ABRE will be able to regulate transcription.

Abscisic Acid↗