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PubMed1993

Isolation of the CCAAT transcription factor subunit EFIA cDNA and a potentially functional EFIA processed pseudogene from Bos taurus: insights into the evolution of the EFIA/dbpB/YB-1 gene family.

The genomic copy multiplicity of the CCAAT transcription complex component enhancer factor I subunit A (EFIA) has been examined. When a mammalian genomic Southern blot was hybridized to a rat EFIA cDNA, a complex pattern consisting of numerous related sequences was found in all the species examined, with Bos taurus being the least complex. An EFIA#1 cDNA from Bos taurus was isolated from a primary lung endothelial cell cDNA library by screening with the 1489-bp rat EFIA cDNA. The deduced bovine EFIA#1 amino acid (aa) sequence is 98% identical to rat EFIA and 100% identical to human EFIA/DbpB/YB-1 family member DNA-binding protein B (DbpB). In addition, a processed EFIA pseudogene from Bos taurus, designated bovine psi EFIA#1, was obtained from a genomic library by screening with a rat EFIA cDNA probe. The bovine psi EFIA#1 gene has an ORF which, if expressed, would encode a 140-aa sequence, with aa 31-140 having 84% identity to bovine EFIA#1. The genomic cloning data indicate that processed pseudogenes are partially responsible for the complexity of the EFIA genomic Southern blots. The phenomenon of 'repeat induced point mutation' (ripping) at bovine psi EFIA#1 gene CpG dinucleotides occurs at a 6.5-fold higher frequency than expected from random mutagenesis. Therefore, ripping is likely to be the mechanism by which the bovine EFIA#1 pseudogene's ectopic recombination potential was inactivated.

Amino Acid Sequence↗

PubMed2006

A homotrimer-heterotrimer switch in Sir2 structure differentiates rDNA and telomeric silencing.

The budding yeast genome contains transcriptionally repressed domains at mating-type and telomeric loci, and within rDNA repeats. Gene silencing at telomeres requires the Silent information regulators Sir2p, Sir3p, and Sir4p, whereas only the Sir2p histone deacetylase is required for rDNA repression. To understand these silencing mechanisms biochemically, we examined the subunit structure of Sir2p-containing complexes. Sir2p alone forms a stable homotrimer, whereas the SIR complex is a heterotrimer containing one copy of each Sir protein. A point mutation in the Sir2p core domain (sir2(P394L)) compromises selectively rDNA repression. This mutation impairs homotrimerization but allows SIR heterotrimer formation. Surprisingly, when sir2(P394L) is coexpressed with wild-type Sir2p, rDNA repression increases and homotrimers form. Furthermore, coexpression of sir2(P394L) and enzymatically inactive sir2(H364Y) allows crosscomplementation of rDNA repression defects. The correlation of genetic and biochemical complementation argues that Sir2p trimerization is physiologically relevant for rDNA silencing.

Amino Acid Sequence↗

PubMed2002

Quantitative proteomics: the copy number of pyruvate dehydrogenase is more than 10(2)-fold lower than that of complex III in human mitochondria.

Pyruvate dehydrogenase (PDH) and complex III are two key protein complexes in mitochondrial metabolic activity. Using a novel quantitative Western blotting method, we find that PDH and complex III exist at a steady-state ratio of 1:100, 1:128 and 1:202 in HeLa cell extracts, fibroblast mitochondria and heart tissue mitochondria, respectively. This difference in stoichiometry is reflected in the immunogold labeling intensities of the two complexes and by the much more sparse distribution of PDH in fluorescence microscopy. In Rho0 fibroblasts there is a 64% reduction of complex III but the concentration of PDH remains the same as wild-type.

Blotting, Western↗

PubMed1998

Nuclear import of polypeptides, polynucleotides and supramolecular complexes.

Nuclear import of macromolecules or particles is a strictly regulated phenomenon that is now understood in some depth at the molecular level. Transport across nuclear membranes is controlled by nuclear pores, with the involvement of cytoplasmic receptors and accessory molecules. Each nuclear pore complex is an assembly of multiple copies of at least 30 distinct proteins, with a total mass of approximately 10(7) kDa. Passive transport of small molecules through nuclear pores is unrestricted, but efficient uptake of macromolecules requires nuclear localization signals, which facilitate the interaction with cytoplasmic receptor proteins. The basic architecture of the nuclear pore and the mechanisms that regulate nuclear import are summarized, in relation to import of endogenous molecules and viruses. Subsequently, the significance of these mechanisms in controlling gene delivery is discussed. Access of DNA to the nucleus is a major barrier to the success of gene therapy, although viruses have evolved mechanisms to exploit the active transport machinery within the host cell. A key step in the future development of non-viral gene therapy will be the design and development of synthetic systems for active delivery of DNA to the nucleus.

Journal Article↗

PubMed1998

Make room for dynein.

Three classes of cytoskeletal motor protein have been identified--myosins, kinesins and dyneins. Together, these proteins are now thought to be responsible for the remarkable variety of movements that occur in eukaryotic cells and that are essential for reproduction and survival. Crystallographic analysis of the myosin and kinesin motor domains at atomic resolution has provided insight into their mechanism of force production. However, because of its relative intractability to molecular manipulation, definition of the dynein motor domain, let alone progress in understanding how it works, has been slower. Evidence now indicates that the microtubule-binding domain of dynein is spatially isolated from the ATPase domain at the tip of a projecting coiled coil. As proposed here, this curious arrangement might serve to accommodate multiple copies of the outsized and functionally complex motor heads on the microtubule surface.

Animals↗

PubMed1984

Elongation factor Tu ternary complex binds to small ribosomal subunits in a functionally active state.

A complex between elongation factor Tu (EF-Tu), GTP, phenylalanyl-tRNA (Phe-tRNA), oligo(uridylic acid) [oligo(U)], and the 30S ribosomal subunit of Escherichia coli has been formed and isolated. Binding of the EF-Tu complex appears to be at the functionally active 30S site, by all biochemical criteria that were examined. The complex can be isolated with 0.25-0.5 copy of EF-Tu bound per ribosome. The binding is dependent upon the presence of both the aminoacyl-tRNA and the cognate messenger RNA. Addition of 50S subunits to the preformed 30S-EF-Tu-GTP-Phe-tRNA-oligo(U) complex ("30S-EF-Tu complex") causes a rapid hydrolysis of GTP. This hydrolysis is coordinated with the formation of 70S ribosomes and the release of EF-Tu. Both the release of EF-Tu and the hydrolysis of GTP are stoichiometric with the amount of added 50S subunits. 70S ribosomes, in contrast to 50S subunits, neither release EF-Tu nor rapidly hydrolyze GTP when added to the 30S-EF-Tu complexes. The inability of 70S ribosomes to react with the 30S-EF-Tu complex argues that the 30S-EF-Tu complex does not dissociate prior to reaction with the 50S subunit. The requirements of the 30S reaction for Phe-tRNA and oligo(U) and the consequences of the addition of 50S subunits resemble the reaction of EF-Tu with 70S ribosomes, although EF-Tu binding to isolated 30S subunits does not occur during the elongation microcycle. This suggests that the EF-Tu ternary complex binds to isolated 30S subunits at the same 30S site that is occupied during ternary complex interaction with the 70S ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗

PubMed2001

Post-transcriptional gene silencing in plum pox virus resistant transgenic European plum containing the plum pox potyvirus coat protein gene.

Transgenic plums containing the plum pox potyvirus coat protein (PPV-CP) gene were inoculated with PPV. Infection was monitored by evaluating symptoms, ELISA, and IC-RT-PCR. Transgenic clone C5 was highly resistant to PPV during four years of testing and displayed characteristics typical of post-transcriptional gene silencing (PTGS), including a high level of transgene transcription in the nucleus, low levels of transgene mRNA in the cytoplasm, a complex multicopy transgene insertion with aberrant copies, and methylation of the silenced PPV-CP transgene. The PPV-CP transgene was also methylated in seedlings of C5 and these seedlings were resistant to PPV. Our results show, for the first time, that PTGS functions as a mechanism for virus resistance in a woody perennial species.

Blotting, Southern↗

PubMed2026

Integrated signatures define mutational processes in prostate cancer.

Prostate cancer follows a long and heterogeneous disease course with incompletely understood aetiology1. Here we dissect the mutational processes shaping the genomes of 959 donors from the Pan Prostate Cancer Group and assess their clinical relevance. By integrating de novo extracted single-base substitution, insertion-deletion and copy-number signatures with six novel complex structural variant signatures, we identify eight integrated mutational footprints (IMFs) that collectively explain the mutational processes in 85% of primary prostate cancer genomes. IMFs were strongly influenced by regional biases in the genome, most prevalently androgen receptor-mediated mutagenesis and replication stress. Four IMFs, present in 37% of primary tumours, were significantly associated with shorter time to metastasis. These included reactive oxygen-species-driven mutagenesis and both canonical and non-canonical homologous recombination deficiency, the latter being enriched in patients of African ancestry. Extending to the metastatic setting, we found that IMFs predicted sensitivity to androgen receptor pathway inhibitors. Taken together, our study delineates the aetiologies and mutational processes that drive the genomic and clinical heterogeneity of prostate cancer, introduces IMFs as a unifying framework, and highlights their potential to improve both risk stratification and biomarker-guided treatment selection.

Journal Article↗

PubMed1994

Transgenic complementation of the hy3 phytochrome B mutation and response to PHYB gene copy number in Arabidopsis.

A recombinant PHYB minigene (mPHYB) consisting of the complete Arabidopsis PHYB cDNA sequence fused to 2.3 kb of upstream PHYB promoter sequence has been introduced into wild-type Arabidopsis and into a strain containing the Bo64 allele of the hy3 mutation. The Bo64 mutant has previously been shown to contain a nonsense mutation in the PHYB coding sequence. Transformation of this strain with the mPHYB gene results in complementation of all of the mutant phenotypic characteristics tested including hypocotyl length and hypocotyl cell size, response to end-of-day far-red light, leaf morphology, chlorophyll level, and flowering time. Presence of the mPHYB transgene in a wild-type genetic background causes exaggeration of this same set of phenotypic characteristics, indicating that these diverse photo-morphogenic responses are sensitive to the copy number of the PHYB gene. The transgene inserts in the Bo64(mPHYB) and WT(mPHYB) lines are shown to be single locus and single copy and the immunologically detectable level of phytochrome B is shown to vary linearly with PHYB gene copy number. These results demonstrate a complex role for phytochrome B in Arabidopsis photo-morphogenesis and suggest that the expression level of this phytochrome gene is an important determinant of the intensity of light-induced plant responses.

Alleles↗

PubMed2002

Penelope retroelements from Drosophila virilis are active after transformation of Drosophila melanogaster.

The Penelope family of retroelements was first described in species of the Drosophila virilis group. Intact elements encode a reverse transcriptase and an endonuclease of the UvrC type, which may play a role in Penelope integration. Penelope is a key element in the induction of D. virilis hybrid dysgenesis, which involves the mobilization of several unrelated families of transposable elements. We here report the successful introduction of Penelope into the germ line of Drosophila melanogaster by P element-mediated transformation with three different constructs. Penelope is actively transcribed in the D. melanogaster genome only in lines transformed with a construct containing a full-length Penelope clone. The transcript is identical to that detected in D. virilis dysgenic hybrids. Most newly transposed Penelope elements have a very complex organization. Significant proliferation of Penelope copy number occurred in some lines during the 24-month period after transformation. The absence of copy number increase with two other constructs suggests that the 5' andor 3' UTRs of Penelope are required for successful transposition in D. melanogaster. No insect retroelement has previously been reported to be actively transcribed and to increase in copy number after interspecific transformation.

Animals↗

PubMed1982

Structure of the mitochondrial F1 ATPase at 9-A resolution.

The soluble portion (F1 ATPase) of the mitochondrial ATP-synthesizing system is a multisubunit enzyme of molecular weight 380,000. It is composed of five different subunits, alpha, beta, gamma, and epsilon. The subunit stoichiometry is not known but there are strong suggestions that it is alpha 3 beta 3 gamma delta epsilon. We have determined the three-dimensional structure of the F1 ATPase of rat liver mitochondria to 9-A resolution by using x-ray diffraction techniques. The molecule appears to be formed by two equivalent halves, each formed by three regions of approximately equal size. These regions form a distorted hexagonal or octahedral arrangement. None of the regions form closed symmetrical trimers in the complex. It is proposed that, if the subunit stoichiometry is alpha 3 beta 3 gamma delta epsilon, the major subunits exist in at least two different environments in the complex. In this arrangement, the different copies of the major subunits are functionally not equivalent. This observation appears to offer a natural explanation of the complicated binding and labeling data of F1 ATPases.

Animals↗

PubMed2006

Neuropsychological differentiation of dementia with Lewy bodies from normal aging and Alzheimer's disease.

We examined the diagnostic utility of selected neuropsychological measures in the differentiation of dementia with Lewy bodies (DLB) from normal aging and Alzheimer's disease (AD). Patients with DLB (n = 87), AD (n = 138), and a group of normal controls (n = 103) were recruited from the Mayo Alzheimer's disease patient registry and Alzheimer's Disease Research Center. Neuropsychological measures shown to have utility in previous studies were included in the analysis. The final multivariate logistic regression model distinguishing DLB from normal controls included Auditory Verbal Learning Test (AVLT) percent retention, Block Design, Trail Making Test-Part A, and Benton Visual Form Discrimination. This model has a sensitivity of 88.6% and specificity of 96.1%. The final multivariate logistic model distinguishing DLB from AD included Trail Making Part A, Boston Naming Test (BNT), AVLT percent retention, and copy of the Rey-Osterrieth Complex Figure. This model had a sensitivity of 83.3% and a specificity of 91.4%. AVLT and BNT had negative coefficients, indicating that lower scores decreased the likelihood of DLB relative to AD. These finding extend prior research suggesting a cognitive profile that can aid in the clinical diagnosis of DLB. Early attention and visual perceptual disturbance suggests DLB, while early impairment in memory and naming suggests AD.

Aged↗

PubMed1987

Molecular cloning of the chicken avidin cDNA.

A cDNA for chicken avidin was identified in a chicken oviduct cDNA library by screening with antibodies and synthetic oligodeoxyribonucleotides. Four recombinant clones were characterized and each contained the sequence of the oligonucleotide probes used in screening. They were capable also of expressing an antigen recognizable by a polyclonal or a mixture of monoclonal antibodies raised against avidin. The longest clone, lambda cAV4, contained the entire coding sequence of avidin along with a signal peptide of 24 amino acids. An avidin mRNA, approximately 700 nucleotides in length, was induced by a single injection of progesterone over a period of twenty four hours. The avidin mRNA was distributed in a tissue-specific manner, since detectable concentration of the mRNA appeared only in the oviduct after stimulation with progesterone alone or with a combination of progesterone and estrogen. No avidin mRNA was detected in the liver or kidney under these conditions. Preliminary results on the genomic complexity of avidin suggest a single copy gene. Isolation of the natural gene for avidin and studies on its regulation now can be initiated using the cDNA probe.

Animals↗

PubMed2006

A reliable method to display authentic DNase I hypersensitive sites at long-ranges in single-copy genes from large genomes.

The study of eukaryotic gene transcription depends on methods to discover distal cis-acting control sequences. Comparative bioinformatics is one powerful strategy to reveal these domains, but still requires conventional wet-bench techniques to elucidate their specificity and function. The DNase I hypersensitivity assay (DHA) is also a method to identify regulatory domains, but can also suggest their function. Technically however, the classical DHA is constrained to mapping gene loci in small increments of approximately 20 kb. This limitation hinders efficient and comprehensive analysis of distal gene regions. Here, we report an improved method termed mega-DHA that extends the range of existing DHAs to facilitate assaying intervals that approach 100 kb. We demonstrate its feasibility for efficient analysis of single-copy genes within a large and complex genome by assaying 230 kb of the human ADAMTS14-perforin-paladin gene cluster in four experiments. The results identify distinct networks of regulatory domains specific to expression of perforin and its two neighboring genes.

Animals↗

PubMed2003

Visual-spatial skills in children after open-heart surgery.

This study was part of a randomized clinical trial comparing the central nervous system effects of the two vital organ-support methods used in infant cardiac surgery: total circulatory arrest and low-flow cardiopulmonary bypass. The extent to which visual-spatial deficits are (1). associated with surgical and perioperative variables, (2). attributable to visual-perceptual, motor control, or metacognitive deficits, and (3). associated with adaptive difficulties at home or school was evaluated. The subjects were 155 8-year-old children with D-transposition of the great arteries who underwent the arterial switch operation before 3 months of age. As part of a comprehensive evaluation, the Rey-Osterrieth Complex Figure (ROCF) was administered. ROCF copy productions were classified as having a Basal Organization Level of 1 (low) or 2 or greater. A five-category clinical rating was also assigned. More than half of the children in the cohort (52%) had copy productions scored at Level 1, more than twice the expected frequency. The risk of having a low score was not associated with vital organ support method or other surgical variables. On the basis of comparisons of the relative fits of nested logistic regression models, poor visual-perceptual abilities were more predictive of having a Level 1 score than either motor control or metacognitive deficits. Children with poor copy production scores had lower mathematics scores, but not lower reading scores or poorer parent and teacher ratings of adaptive competence. The percentage of children receiving remedial school services was associated with ROCF clinical rating, ranging from 58% in the worst category to 8% in the best category. Visual-spatial deficits are common among children after infant heart surgery and seem to reflect visual-perceptual rather than motor control or metacognitive deficits. In addition, these deficits do not seem to be clearly associated with the intraoperative methods or postoperative events evaluated.

Cardiac Surgical Procedures↗

PubMedDate not supplied

Evolutionarily conserved low copy repeats (LCRs) in 22q11 mediate deletions, duplications, translocations, and genomic instability: an update and literature review.

Several constitutional rearrangements, including deletions, duplications, and translocations, are associated with 22q11.2. These rearrangements give rise to a variety of genomic disorders, including DiGeorge, velocardiofacial, and conotruncal anomaly face syndromes (DGS/VCFS/CAFS), cat eye syndrome (CES), and the supernumerary der(22)t(11;22) syndrome associated with the recurrent t(11;22). Chromosome 22-specific duplications or low copy repeats (LCRs) have been directly implicated in the chromosomal rearrangements associated with 22q11.2. Extensive sequence analysis of the different copies of 22q11 LCRs suggests a complex organization. Examination of their evolutionary origin suggests that the duplications in 22q11.2 may predate the divergence of New World monkeys 40 million years ago. Based on the current data, a number of models are proposed to explain the LCR-mediated constitutional rearrangements of 22q11.2.

Abnormalities, Multiple↗

PubMed2026

Mitochondrial Haplotype Shapes the Trajectory of Ovarian Aging in Genetically Heterogeneous Rats.

Ovarian aging leads to permanent reproductive senescence and systemic hormonal changes that predispose women to age-associated comorbidities. Despite these observations, the intrinsic mechanisms driving age-related ovarian decline are poorly defined. Mitochondrial DNA (mtDNA) mutations and instability are strongly associated with aging; however, it remains unknown if naturally occurring mitochondrial genetic variation influences the trajectory of ovarian aging. To address this, we compared two genetically heterogeneous rat cohorts (OKC-HETB and OKC-HETW) that differ in mitochondrial haplotype on a randomized but equivalently distributed nuclear background. The OKC-HETW haplotype was associated with accelerated loss of primordial follicles and pathological remodeling marked by fibrosis, macrophage infiltration, and multinucleated giant cells. These tissue-level pathologies were paralleled by mitochondrial dysfunction, characterized by decreased respiratory complex activity, ATP production, and mtDNA copy number. Mechanistically, we identified a haplotype-specific defect in mitochondrial genome maintenance. Although TFAM expression was normal, and total TFAM protein was elevated, OKC-HETW ovaries showed reduced mitochondrial TFAM abundance, TFAM-mtDNA binding, and TOMM20, suggesting that impaired TOMM20-mediated import is associated with compromised mitochondrial genomic stability. Longitudinal transcriptomic and proteomic analyses further indicate that mitochondrial haplotype influences the rate of ovarian aging, with OKC-HETW ovaries showing accelerated activation of inflammatory and fibrotic pathways alongside suppressed proteostasis and mitochondrial function. These defects corresponded to impairments in ovulation and a trend toward worsening oocyte quality. Collectively, our findings identify mitochondrial haplotype as a heritable modifier of ovarian aging rate that acts in concert with the nuclear genome, and a putative target for preserving ovarian function and female healthspan.

Animals↗

PubMed2005

High-level long-term white blood cell microchimerism after transfusion of leukoreduced blood components to patients resuscitated after severe traumatic injury.

BACKGROUND: Long-term white blood cell (WBC) microchimerism (MC), of at least 2 years, has been reported in trauma patients receiving fresh nonleukoreduced (non-LR) blood. It is unknown, however, whether this occurs with LR blood products that are nearly devoid of WBCs. Twenty-seven patients transfused with LR and non-LR blood products were studied after severe traumatic injury. A secondary aim was to explore donor-recipient mixed lymphocyte reactivity in vitro. STUDY DESIGN AND METHODS: To quantify MC, allele-specific real-time polymerase chain reaction assays were developed targeting HLA Class II sequence polymorphisms. Extensive validation showed that these assays reliably detect a single copy of target sequence in a complex allogeneic background without false positivity. RESULTS: At a median follow-up of 26 months (range, 24-39 months), long-term MC was observed in 3 of 20 patients (15%) who received non-LR blood products and 2 of 7 (29%) who received LR blood products. The maximum MC ranged from 0.40 to 4.90 percent of circulating WBCs and appeared, by Class II genotype analysis, to be attributable to a single donor. CONCLUSION: It is concluded that robust levels of long-term MC, apparently traceable to a single donor, occur at similar frequency despite leukoreduction of transfused blood products. Exploratory analysis of donor-recipient mixed lymphocyte reactivity suggests that long-term MC may require a state of bidirectional tolerance before transfusion.

Blood Component Transfusion↗