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PubMed1983

Cloning of total mRNA populations from adult and embryonic mice.

Total clone banks of cDNAs synthesized from poly(A)-RNA obtained from three stages of the developing mouse were constructed. The stages chosen were 13-day-old embryo, neonatal, and fully grown adult. To have as complete a bank as possible, large numbers of individual clones were generated approximately 400,000 for the 13th day embryo and neonatal mouse and approximately 610,000 for the adult bank. In each case the clone bank was constructed by inserting double stranded cDNA into the PstI site of pBR322 by the "G-C tailing" method. Sequences cloned in this way could be separated from the plasmid host DNA by treatment of the resultant total chimeric plasmid population with PstI. Aliquots of the cloned cDNA material were labeled with 32P by "nick translation" using Escherichia coli DNA polymerase I for the preparation of hybridization probes. Back-hybridization of these probes to the total clone banks allowed the determination of the sequence diversity among the above three very different developmental stages. The use of such clone banks should allow the identification of developmental stage specific mRNAs.

Age Factors↗
PubMed1991

Temperature-sensitive variants of Saccharomyces cerevisiae iso-1-cytochrome c produced by random mutagenesis of codons 43 to 54.

In vitro random mutagenesis within the CYC1 gene from the yeast Saccharomyces cerevisiae was used to produce a library of mutants encompassing codons 43 to 54 of iso-1-cytochrome c. This region consists of an evolutionarily conserved structure within an evolutionarily diverse sequence. The library, on a low-copy-number yeast shuttle phagemid, was introduced into a yeast strain lacking cytochrome c. The ability of transformants harboring a functional cytochrome c to grow on the non-fermentable carbon source glycerol at 30 degrees C and 37 degrees C was used to determine the phenotype of nearly 1000 transformants. Approximately 90% of the missense mutants present in the library give rise to the wild-type phenotype, 7% result in the temperature-sensitive (Cycts) phenotype, and 3% give rise to the non-functional (Cyc-) phenotype. Phagemids from 20 Cycts and 30 Cyc- transformants were subjected to DNA sequence analysis. All the mutations occur within the targeted region. One-third of the mutants from Cyc- transformants and all the mutants from Cycts transformants are missense mutants. The remaining mutants from Cyc- transformants are nonsense or frame-shift mutants. Missense mutations within the codons for Gly45, Tyr46, Thr49, Asn52 or Ile53 alone are sufficient to produce temperature-sensitive behavior both in vivo and in the variant proteins. The deduced amino acid substitutions correlate remarkably well with side-chain dynamics, secondary structure and tertiary structure of the wild-type protein.

Base Sequence↗
PubMed1981

Ageing studies in rat liver. I. Complexity of RNA from two to ten months of age.

A highly sensitive nucleic acid hybridization assay was used to compare the extent of nonrepetitive DNA transcription in rat liver between the ages of two and ten months. The basic approach consisted on initially purifying the DNA expressed in liver at these ages and then using it in reactions with homologous and heterologous RNAs. Such experiments failed to reveal any differences in nonrepetitive DNA transcription as a function of age. The possibility was also explored that there might be an age-associated variation in the proportion of the total RNA complexity attributable to poly(A+) RNA. These experiments, too, were negative in that the poly(A+) RNA was found in all cases to account for approximately 50% of the total sequence diversity. Overall, these data strongly suggest that ageing is not accompanied by a steady, progressive change in the regions of the genome transcribed, either quantitatively or qualitatively.

Animals↗
PubMed1979

Site-specific cleavage of DNA by E. coli DNA gyrase.

E. coli DNA gyrase, which catalyzes the supercoiling of DNA, cleaves DNA site-specifically when oxolinic acid and sodium dodecylsulfate are added to the reaction. We studied the structure of the gyrasecleaved DNA because of its implications for the reaction mechanism and biological role of gyrase. Gyrase made a staggered cut, creating DNA termini with a free 3' hydroxyl and a 5' extension that provided a template primer for DNA polymerase. The cleaved DNA was resistant to labeling with T4 polynucleotide kinase even after treatment with proteinase K. Thus the denatured enzyme that remains attached to cleaved DNA is covalently bonded to both 5' terminal extensions. The 5' extensions of many gyrase cleavage fragments from phi X174, SV40 and Col E1 DNA were partially sequenced using repair with E. coli DNA polymerase I. No unique sequence existed within the cohesive ends, but G was the predominant first base incorporated by DNA polymerase I. The cohesive and sequences of four gyrase sites were determined, and they demonstrated a four base 5' extension. The dinucleotide TG, straddling the gyrase cut on one DNA strand, provided the only common bases within a 100 bp region surrounding the cleavage sites. Analysis of other cleavage fragments showed that cutting between a TG doublet is common to most, or all, gyrase cleavages. Other bases common to some of the sequenced sites were clustered nonrandomly around the TG doublet, and may be variable components of the cleavage sequence. This diverse recognition sequence with common elements is a pattern shared with several other specific nucleic acid-protein interactions.

Base Sequence↗
PubMed1986

Identification of visceral Leishmania species with cloned sequences of kinetoplast DNA.

Several efforts have been made in order to develop more precise and sensitive methods in the identification of Leishmania parasites. We report here the identification of cloned subfragments of minicircle kinetoplast DNA (kDNA) isolated from L. donovani, WR352, which show different taxonomic specificities. Analysis of these fragments demonstrates a significant sequence diversity within the kDNA minicircle. For example, one cloned fragment was found to be present in all visceral Leishmania species tested, but was not present in any of the cutaneous Leishmania species. Another cloned fragment was only found in the strain from which it had been derived, and was not present in any of the other strains tested. In similar experiments with the New World visceral leishmanias (L. chagasi, WR518) several different cloned kDNA fragments were found to react with all of isolates of the L. chagasi tested, but not with any cutaneous Leishmania species, either from the Old World or the New World. It is of interest to note that these cloned L. chagasi kDNA fragments reacted with isolates of African visceral Leishmania species but not with isolates from India.

Animals↗
PubMed1992

Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.

Adolescent↗
PubMed2004

A method for the non-covalent immobilization of heparin to surfaces.

The interaction of heparan sulfate (HS) with specific proteins facilitates a wide range of fundamental biological processes including cellular proliferation and differentiation, tissue homeostasis, and viral pathogenesis. This multiplicity of function arises through sequence diversity within the HS chain. Heparin, which is very similar in structure to the sulfated regions of HS, is an excellent model for studying HS-protein interactions. The development of high-throughput enzyme-linked immunosorbent-like assays using surface-immobilized heparin has been hindered by the inability of this glycosaminoglycan to adhere to microtiter surfaces. Here we report the passive noncovalent adsorption of heparin onto microtiter wells following their treatment by plasma polymerization; there was no detectable binding of functional heparin onto untreated plates. Heparin immobilized in this way was able to interact with four different heparin-binding proteins tested, i.e., TSG-6, chemokines IL-8 and KC, and complement factor H. Heparin preparations ranging in size from high molecular weight to a defined decasaccharide could be adsorbed onto these plates in a functionally active form. Since plasma polymerization is possible for virtually any surface, this technique is likely to be of general use in the identification and characterization of heparin/HS-binding proteins in a wide range of applications.

Allylamine↗
PubMed2005

Biotinylation of heat shock protein 70 induces RANTES production in HEK293 cells in a CD40-independent pathway.

Biotinylated proteins and peptides have been used as popular ligands for characterization of cell surface receptors by a variety of methods including flow cytometry. The number and the location of biotin moieties incorporated could alter the structural and physicochemical properties of ligands, although biotin is thought to be such a small molecule (244Da) that it is capable of being conjugated to most proteins without affecting their activity. Here, we demonstrate that the biotinylated HSP70 molecule via primary amines bound to epithelium-like HEK 293 cells in a saturable manner whereas the unlabeled counterparts of HSP70 other than mouse Hsp72 do not. This binding was not competed by either HSP70 or the biotin entity itself. Interestingly, the biotinylated HSP70 also elicited the production of CC-chemokine RANTES independent of CD40 signaling. This response occurred regardless of sequence diversity of HSP70 derived from different species, and neither the biotinylated ovalbumin nor the unlabeled HSP70 cross-linked with a biotinylated protein stimulated a significant level of RANTES production which was induced by biotinylated HSP70 itself. Our findings suggest that modification of HSP70 such as biotinylation may function as a biological alarm signal in the innate immune system.

Biotin↗
PubMed2005

beta-Peptides as inhibitors of protein-protein interactions.

We became interested several years ago in exploring whether 14-helical beta-peptide foldamers could bind protein surfaces and inhibit protein-protein interactions, and if so, whether their affinities and specificities would compare favorably with those of natural or miniature proteins. This exploration was complicated initially by the absence of a suitable beta-peptide scaffold, one that possessed a well-defined 14-helical structure in water and tolerated the diverse sequence variation required to generate high-affinity protein surface ligands. In this perspective, we describe our approach to the design of adaptable beta-peptide scaffolds with high levels of 14-helix structure in water, track the subsequent development of 14-helical beta-peptide protein-protein interaction inhibitors, and examine the potential of this strategy for targeting other therapeutically important proteins.

Peptides↗
PubMed2006

The why and how of thymocyte negative selection.

The generation of T cell receptor (TCR) sequence diversity is the strength of adaptive immunity, yet is also the Achilles' heel. To purge highly self-reactive T cells from the immune system, generation of diversity has coevolved with a mechanism of negative selection. Recent studies have revealed new insights addressing the why and how of negative selection by examining situations in which negative selection has failed in human and animals models of autoimmunity. Both thymocyte extrinsic and intrinsic mechanisms are required to restrict the TCR repertoire to a non-autoreactive set. Negative selection also ensures that T cells emerge with receptors that are focussed on the peptide moiety of MHC-peptide complexes.

Animals↗
PubMed2026

Structure-informed theoretical modeling defines principles governing avidity in bivalent protein interactions.

In signaling cascades, signaling proteins often encode multiple domains or motifs, which presents the possibility for avidity -- where multivalent binding drastically increases interaction strength and duration. However, predicting and validating multivalent interactions that interact with avidity is a challenge. Here, we integrate mechanistic modeling, structure-based analysis, and experimental approaches as a framework for defining the conditions under which avidity plays a role. We explore the tandem SH2 domain family of interactions with bisphosphorylated partners as a multivalent archetype, which encompasses key secondary messengers in tyrosine kinase signaling networks. Theoretical modeling suggests that maximum avidity occurs with closely spaced tyrosine phosphorylation sites combined with moderate monovalent affinities - exactly around the innate range of SH2 domain affinity - or with phosphorylation sites separated by sufficiently flexible linkers. Surprisingly, despite sequence diversity, structure-based analysis showed relatively conserved three-dimensional spacing between SH2 domains across all tandem SH2 families, which we corroborate experimentally, suggesting evolutionary optimization for avidity interactions. The combination of structure-based analysis of domain spacing with available monovalent experimental data appears, along with iterative experimental refinement of biophysical parameters, can identify high affinity interactions of tandem SH2 domain recruitment to the EGFR C-terminal tail. Using these principles, we extended bivalent predictions into the full phosphoproteome space and structural parameterization of other partners of SH2 domain binding, providing resources and methods for more rapid expansion of bivalent analysis. These approaches lay the groundwork for larger utility in multivalent prediction and testing to help better understand protein interactions that drive cell signaling.

BLI↗
PubMed2005

Is there an association between ocular adnexal lymphoma and infection with Chlamydia psittaci? The University of Rochester experience.

Various subsets of extranodal marginal zone lymphomas of mucosa-associated lymphoid tissues (MALT lymphomas) have been associated with infectious organisms. Most notable of these is the association of gastric MALT lymphomas with Helicobacter pylori infection. In a recent publication Ferreri et al. [Ferreri AJ, Guidoboni M, Ponzoni M, De Conciliis C, Dell'Oro S, Fleischhauer K, et al. Evidence for an association between Chlamydia psittaci and ocular adnexal lymphomas. J Natl Cancer Inst 2004;96:586-94] reported the presence of C. psittaci DNA in 80% of 40 ocular adnexal lymphomas. Similar to the gastric MALT lymphoma data, a subset of these patients responded well to antibiotic treatment. We analyzed a set of ocular adnexal lymphomas and benign (non-neoplastic) lesions for evidence of C. psittaci DNA in patients from New York State. No evidence of C. psittaci DNA was seen in seven MALT-type ocular adnexal lymphomas, four non-MALT ocular lymphomas, one Langerhans histiocytosis, and five reactive lymphoproliferations. We eliminated several possible reasons that would cause our study to fail to find C. psittaci DNA, including the presence of PCR inhibitors, inadequate template DNA, and sequence diversity in the target region in C. psittaci. The positive data were based primarily on patients from Italy, while our study involved only patients living in the Northeastern United States. This would suggest possible geographic differences in the etiology of ocular adnexal lymphomas.

Adolescent↗
PubMed2004

Insulin degrading enzyme is expressed in the human cerebrovascular endothelium and in cultured human cerebrovascular endothelial cells.

Insulin degrading enzyme (IDE) is found in the cytosol, peroxisomes and plasma membrane of many cells. Although it preferentially cleaves insulin it can also cleave many other small proteins with diverse sequences including the monomeric form of the amyloid beta peptide (A beta). In the brain, IDE has been reported to be expressed predominantly in neurons. In this study, IDE expression was detected in cultured human cerebrovascular endothelial cells. Using laser capture microdissection followed by PCR analysis, it was found that IDE mRNA is expressed in human brain blood vessels. Using immunofluorescence and multiphoton microscopy IDE was localized to the endothelium of the cerebrovascular blood vessels in human.

Cells, Cultured↗
PubMed2006

Proline and glycine control protein self-organization into elastomeric or amyloid fibrils.

Elastin provides extensible tissues, including arteries and skin, with the propensity for elastic recoil, whereas amyloid fibrils are associated with tissue-degenerative diseases, such as Alzheimer's. Although both elastin-like and amyloid-like materials result from the self-organization of proteins into fibrils, the molecular basis of their differing physical properties is poorly understood. Using molecular simulations of monomeric and aggregated states, we demonstrate that elastin-like and amyloid-like peptides are separable on the basis of backbone hydration and peptide-peptide hydrogen bonding. The analysis of diverse sequences, including those of elastin, amyloids, spider silks, wheat gluten, and insect resilin, reveals a threshold in proline and glycine composition above which amyloid formation is impeded and elastomeric properties become apparent. The predictive capacity of this threshold is confirmed by the self-assembly of recombinant peptides into either amyloid or elastin-like fibrils. Our findings support a unified model of protein aggregation in which hydration and conformational disorder are fundamental requirements for elastomeric function.

Alzheimer Disease↗
PubMed2007

Methods for linkage disequilibrium mapping in crops.

Linkage disequilibrium (LD) mapping in plants detects and locates quantitative trait loci (QTL) by the strength of the correlation between a trait and a marker. It offers greater precision in QTL location than family-based linkage analysis and should therefore lead to more efficient marker-assisted selection, facilitate gene discovery and help to meet the challenge of connecting sequence diversity with heritable phenotypic differences. Unlike family-based linkage analysis, LD mapping does not require family or pedigree information and can be applied to a range of experimental and non-experimental populations. However, care must be taken during analysis to control for the increased rate of false positive results arising from population structure and variety interrelationships. In this review, we discuss how suitable the recently developed alternative methods of LD mapping are for crops.

Chromosome Mapping↗
PubMed2005

Genome analysis of Cryphonectria hypovirus 4, the most common hypovirus species in North America.

Many different viruses that reduce virulence and alter the phenotype to varying extents have been identified in the chestnut blight fungus Cryphonectria parasitica. Most viruses identified in this fungus fall within the Hypoviridae family of positive-sense RNA viruses, which contains one genus and four species. Different species predominate in different geographic locations in chestnut-growing areas around the world. In this paper, we describe the genome organization and some variants of Cryphonectria hypovirus 4 (CHV-4), the species most commonly found in eastern North America. CHV-4 is distinguished from other hypoviruses by having little effect on fungal virulence and colony morphology. The 9.1-kb genome of strain CHV-4/SR2 is the smallest of any member of the family characterized to date. Like the recently characterized species CHV-3, a single ORF was predicted from deduced translations of CHV-4/SR2. Sequence analysis revealed the presence of a putative glucosyltransferase domain in both CHV-4 and in CHV-3, but no such homolog was detected in the more thoroughly examined CHV-1 or in CHV-2. Alignments with 8 other CHV-4 isolates from different regions of eastern North America revealed sequence diversity within the species and the likelihood that RNA recombination has led to this diversity.

Ascomycota↗
PubMed2026

Distinct YY dinucleotide periodicity in adeno-associated virus DNA.

Dinucleotide periodicity is a hallmark of genome organization, yet its role in single-stranded (ss)DNA viruses remains poorly understood. Here, we systematically analyzed dinucleotide spacing patterns in adeno-associated virus (AAV) genomes and other viruses. Across 13 primate AAV serotypes, we identified a pronounced and highly conserved ∼15-bp periodicity specific to pyrimidine-pyrimidine (YY) dinucleotides and their reverse complements (RR). Comparative analyses across >25,000 viral sequences demonstrate that this 15-bp YY/RR periodicity is unique to the genus Dependoparvovirus and absent from other ssDNA viruses, satellite viruses, and helper viruses, which predominantly exhibit canonical ∼10- to 11-bp periodicities. Upon disruption of the YY/RR pattern using DNA family shuffling of AAV capsid genes, and subsequent iterative selection for viral production or cell entry, we found that the pattern is under positive selection. Selected sequences display increased periodicity alongside reduced sequence diversity, supporting a functional role for this genomic feature. Finally, engineered recombinant AAV genomes containing YY periodic motifs exhibit enhanced production and, for some designs, improved transduction efficiency, demonstrating that YY periodicity can modulate viral replication and infectivity. Our findings uncover a unique DNA-encoded signal in dependoparvoviruses that contributes to AAV fitness, expands our knowledge of virus biology, and has implications for vector engineering.

Dependovirus↗
PubMed2003

General function of N-terminal propeptide on assisting protein folding and inhibiting catalytic activity based on observations with a chimeric thermolysin-like protease.

Pro-aminopeptidase processing protease (PA protease) is a thermolysin-like metalloprotease produced by Aeromonas caviae T-64. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of PA protease and shows inhibitory activity toward its cognate mature enzyme. Moreover, the N-terminal propeptide strongly inhibits the autoprocessing of the C-terminal propeptide by forming a complex with the folded intermediate pro-PA protease containing the C-terminal propeptide (MC). In order to investigate the structural determinants within the N-terminal propeptide that play a role in the folding, processing, and enzyme inhibition of PA protease, we constructed a chimeric pro-PA protease by replacing the N-terminal propeptide with that of vibriolysin, a homologue of PA protease. Our results indicated that, although the N-terminal propeptide of vibriolysin shares only 36% identity with that of PA protease, it assists the refolding of MC, inhibits the folded MC to process its C-terminal propeptide, and shows a stronger inhibitory activity toward the mature PA protease than that of PA protease. These results suggest that the N-terminal propeptide domains in these thermolysin-like proteases may have similar functions, in spite of their primary sequence diversity. In addition, the conserved regions in the N-terminal propeptides of PA protease and vibriolysin may be essential for the functions of the N-terminal propeptide.

Aeromonas↗