Fluorescence in situ hybridization analysis of keratinocyte growth factor gene amplification and dispersion in evolution of great apes and humans

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family. Portions of the gene encoding KGF were amplified during primate evolution and are present in multiple nonprocessed copies in the human genome. Nucleotide analysis of a representative sampling of these KGF-like sequences indicated that they were at least 95% identical to corresponding regions of the KGF gene. To localize these sequences to specific chromosomal sites in human and higher primates, we used fluorescence in situ hybridization. In human, using a cosmid probe encoding KGF exon 1, we assigned the location of the KGF gene to chromosome 15q15–21.1. In addition, copies of KGF-like sequences hybridizing only with a cosmid probe encoding exons 2 and 3 were localized to dispersed sites on chromosome 2q21, 9p11, 9q12–13, 18p11, 18q11, 21q11, and 21q21.1. The distribution of KGF-like sequences suggests a role for alphoid DNA in their amplification and dispersion. In chimpanzee, KGF-like sequences were observed at five chromosomal sites, which were each homologous to sites in human, while in gorilla, a subset of four of these homologous sites was identified; in orangutan two sites were identified, while gibbon exhibited only a single site. The chromosomal localization of KGF sequences in human and great ape genomes indicates that amplification and dispersion occurred in multiple discrete steps, with initial KGF gene duplication and dispersion taking place in gibbon and involving loci corresponding to human chromosomes 15 and 21. These findings support the concept of a closer evolutionary relationship of human and chimpanzee and a possible selective pressure for such dispersion during the evolution of higher primates.

Induction of inhibitory factor κBα mRNA in the central nervous system after peripheral lipopolysaccharide administration: An in situ hybridization histochemistry study in the rat

In this study we investigate the mRNA expression of inhibitory factor κBα (IκBα) in cells of the rat brain induced by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). IκB controls the activity of nuclear factor κB, which regulates the transcription of many immune signal molecules. The detection of IκB induction, therefore, would reveal the extent and the cellular location of brain-derived immune molecules in response to peripheral immune challenges. Low levels of IκBα mRNA were found in the large blood vessels and in circumventricular organs (CVOs) of saline-injected control animals. After an i.p. LPS injection (2.5 mg/kg), dramatic induction of IκBα mRNA occurred in four spatio-temporal patterns. Induced signals were first detected at 0.5 hr in the lumen of large blood vessels and in blood vessels of the choroid plexus and CVOs. Second, at 1–2 hr, labeling dramatically increased in the CVOs and choroid plexus and spread to small vascular and glial cells throughout the entire brain; these responses peaked at 2 hr and declined thereafter. Third, cells of the meninges became activated at 2 hr and persisted until 12 hr after the LPS injection. Finally, only at 12 hr, induced signals were present in ventricular ependyma. Thus, IκBα mRNA is induced in brain after peripheral LPS injection, beginning in cells lining the blood side of the blood–brain barrier and progressing to cells inside brain. The spatiotemporal patterns suggest that cells of the blood–brain barrier synthesize immune signal molecules to activate cells inside the central nervous system in response to peripheral LPS. The cerebrospinal fluid appears to be a conduit for these signal molecules.

PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.

A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window

The only treatment of patients with acute ischemic stroke is thrombolytic therapy, which benefits only a fraction of stroke patients. Both human and experimental studies indicate that ischemic stroke involves secondary inflammation that significantly contributes to the outcome after ischemic insult. Minocycline is a semisynthetic second-generation tetracycline that exerts antiinflammatory effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against focal brain ischemia with a wide therapeutic window. Using a rat model of transient middle cerebral artery occlusion, we show that daily treatment with minocycline reduces cortical infarction volume by 76 ± 22% when the treatment is started 12 h before ischemia and by 63 ± 35% when started even 4 h after the onset of ischemia. The treatment inhibits morphological activation of microglia in the area adjacent to the infarction, inhibits induction of IL-1β-converting enzyme, and reduces cyclooxygenase-2 expression and prostaglandin E2 production. Minocycline had no effect on astrogliosis or spreading depression, a wave of ionic transients thought to contribute to enlargement of cortical infarction. Treatment with minocycline may act directly on brain cells, because cultured primary neurons were also salvaged from glutamate toxicity. Minocycline may represent a prototype of an antiinflammatory compound that provides protection against ischemic stroke and has a clinically relevant therapeutic window.

Fluorescence-intensity distribution analysis and its application in biomolecular detection technology

A methodology, fluorescence-intensity distribution analysis, has been developed for confocal microscopy studies in which the fluorescence intensity of a sample with a heterogeneous brightness profile is monitored. An adjustable formula, modeling the spatial brightness distribution, and the technique of generating functions for calculation of theoretical photon count number distributions serve as the two cornerstones of the methodology. The method permits the simultaneous determination of concentrations and specific brightness values of a number of individual fluorescent species in solution. Accordingly, we present an extremely sensitive tool to monitor the interaction of fluorescently labeled molecules or other microparticles with their respective biological counterparts that should find a wide application in life sciences, medicine, and drug discovery. Its potential is demonstrated by studying the hybridization of 5′-(6-carboxytetramethylrhodamine)-labeled and nonlabeled complementary oligonucleotides and the subsequent cleavage of the DNA hybrids by restriction enzymes.

A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families

Systemic lupus erythematosus (SLE) is an autoimmune multisystem inflammatory disease characterized by the production of pathogenic autoantibodies. Previous genetic studies have suggested associations with HLA Class II alleles, complement gene deficiencies, and Fc receptor polymorphisms; however, it is likely that other genes contribute to SLE susceptibility and pathogenesis. Here, we report the results of a genome-wide microsatellite marker screen in 105 SLE sib-pair families. By using multipoint nonparametric methods, the strongest evidence for linkage was found near the HLA locus (6p11-p21) [D6S257, logarithm of odds (lod) = 3.90, P = 0.000011] and at three additional regions: 16q13 (D16S415, lod = 3.64, P = 0.000022), 14q21–23 (D14S276, lod = 2.81, P = 0.00016), and 20p12 (D20S186, lod = 2.62, P = 0.00025). Another nine regions (1p36, 1p13, 1q42, 2p15, 2q21–33, 3cent-q11, 4q28, 11p15, and 15q26) were identified with lod scores ≥1.00. These data support the hypothesis that multiple genes, including one in the HLA region, influence susceptibility to human SLE.

Identification of transforming growth factor-β- regulated genes in Caenorhabditis elegans by differential hybridization of arrayed cDNAs

Members of the transforming growth factor-β family play critical roles in body patterning, in both vertebrates and invertebrates. One transforming growth factor-β-related gene, dbl-1, has been shown to regulate body length and male ray patterning in Caenorhabditis elegans. We screened arrayed cDNAs to identify downstream target genes for the DBL-1 signaling by using differential hybridization. C. elegans cDNAs representing 7,584 independent genes were arrayed on a nylon membrane at high density and hybridized with 33P-labeled DNA probes synthesized from the mRNAs of wild-type, dbl-1, sma-2, and lon-2 worms. Signals for all the spots representing hybridized DNA were quantified and compared among strains. The screening identified 22 and 2 clones, which were positively and negatively regulated, respectively, by the DBL-1 signal. Northern hybridization confirmed the expression profiles of most of the clones, indicating good reliability of the differential hybridization using arrayed cDNAs. In situ hybridization analysis revealed the spatial and temporal expression patterns of each clone and showed that at least four genes, including the gene for the type I receptor for DBL-1, sma-6, were transcriptionally regulated by the DBL-1 signal.

tRNA Nuclear Export in Saccharomyces cerevisiae: In Situ Hybridization Analysis

To understand the factors specifically affecting tRNA nuclear export, we adapted in situ hybridization procedures to locate endogenous levels of individual tRNA families in wild-type and mutant yeast cells. Our studies of tRNAs encoded by genes lacking introns show that nucleoporin Nup116p affects both poly(A) RNA and tRNA export, whereas Nup159p affects only poly(A) RNA export. Los1p is similar to exportin-t, which facilitates vertebrate tRNA export. A los1 deletion mutation affects tRNA but not poly(A) RNA export. The data support the notion that Los1p and exportin-t are functional homologues. Because LOS1 is nonessential, tRNA export in vertebrate and yeast cells likely involves factors in addition to exportin-t. Mutation of RNA1, which encodes RanGAP, causes nuclear accumulation of tRNAs and poly(A) RNA. Many yeast mutants, including those with the rna1-1 mutation, affect both pre-tRNA splicing and RNA export. Our studies of the location of intron-containing pre-tRNAs in the rna1-1 mutant rule out the possibility that this results from tRNA export occurring before splicing. Our results also argue against inappropriate subnuclear compartmentalization causing defects in pre-tRNA splicing. Rather, the data support “feedback” of nucleus/cytosol exchange to the pre-tRNA splicing machinery.

Peptide nucleic acid pre-gel hybridization: An alternative to Southern hybridization

We have found that it is possible to use labeled peptide nucleic acid (PNA)-oligomers as probes in pre-gel hybridization experiments, as an alternative for Southern hybridization. In this technique, the PNA probe is hybridized to a denatured DNA sample at low ionic strength and the mixture is loaded directly on to an electrophoresis system for size separation. Ensuing gel electrophoresis separates the single-stranded DNA fragments by length. The neutral backbone of PNA allows for hybridization at low ionic strength and imparts very low mobility to excess PNA. Detection of the bound PNA is possible by direct fluorescence detection with capillary electrophoresis, or the DNA/PNA hybrids can be blotted onto a membrane and detected with standard chemiluminescent techniques. Efficient single bp discrimination was achieved routinely using both capillary and slab-gel electrophoresis.

Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA

An approach was developed for the quantification of subtle gains and losses of genomic DNA. The approach relies on a process called molecular combing. Molecular combing consists of the extension and alignment of purified molecules of genomic DNA on a glass coverslip. It has the advantage that a large number of genomes can be combed per coverslip, which allows for a statistically adequate number of measurements to be made on the combed DNA. Consequently, a high-resolution approach to mapping and quantifying genomic alterations is possible. The approach consists of applying fluorescence hybridization to the combed DNA by using probes to identify the amplified region. Measurements then are made on the linear hybridization signals to ascertain the region's exact size. The reliability of the approach first was tested for low copy number amplifications by determining the copy number of chromosome 21 in a normal and trisomy 21 cell line. It then was tested for high copy number amplifications by quantifying the copy number of an oncogene amplified in the tumor cell line GTL-16. These results demonstrate that a wide range of amplifications can be accurately and reliably quantified. The sensitivity and resolution of the approach likewise was assessed by determining the copy number of a single allele (160 kb) alteration.

A host-specific function is required for ligation of a wide variety of ribozyme-processed RNAs

Hepatitis δ virus (HDV) replicates its circular RNA genome via a rolling circle mechanism. During this process, cis-acting ribozymes cleave adjacent upstream sequences and thereby resolve replication intermediates to unit-length RNA. The subsequent ligation of these 5′OH and 2′,3′-cyclic phosphate termini to form circular RNA is an essential step in the life cycle of the virus. Here we present evidence for the involvement of a host activity in the ligation of HDV RNA. We used both HDV and hammerhead ribozymes to generate a panel of HDV and non-HDV RNA substrates that bear 5′ hydroxyl and 2′,3′- cyclic phosphate termini. We found that ligation of these substrates occurred in host cells, but not in vitro or in Escherichia coli. The host-specific ligation activity was capable of joining RNA in both bimolecular and intramolecular reactions and functioned in a sequence-independent manner. We conclude that mammalian cells contain a default pathway that efficiently circularizes ribozyme processed RNAs. This pathway could be exploited in the delivery of stable antisense and decoy RNA to the nucleus.

Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 × 10−7 cm2/s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (≤1 × 10−7 cm2/s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 × 10−7 cm2/s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.

Singular value decomposition for genome-wide expression data processing and modeling

We describe the use of singular value decomposition in transforming genome-wide expression data from genes × arrays space to reduced diagonalized “eigengenes” × “eigenarrays” space, where the eigengenes (or eigenarrays) are unique orthonormal superpositions of the genes (or arrays). Normalizing the data by filtering out the eigengenes (and eigenarrays) that are inferred to represent noise or experimental artifacts enables meaningful comparison of the expression of different genes across different arrays in different experiments. Sorting the data according to the eigengenes and eigenarrays gives a global picture of the dynamics of gene expression, in which individual genes and arrays appear to be classified into groups of similar regulation and function, or similar cellular state and biological phenotype, respectively. After normalization and sorting, the significant eigengenes and eigenarrays can be associated with observed genome-wide effects of regulators, or with measured samples, in which these regulators are overactive or underactive, respectively.

New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study

Rheumatoid arthritis (RA), the most common autoimmune disease, is associated in families with other autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM). Its genetic component has been suggested by familial aggregation (λs = 5), twin studies, and segregation analysis. HLA, which is the only susceptibility locus known, has been estimated to account for one-third of this component. The aim of this paper was to identify new RA loci. A genome scan was performed with 114 European Caucasian RA sib pairs from 97 nuclear families. Linkage was significant only for HLA (P < 2.5⋅10−5) and nominal for 19 markers in 14 other regions (P < 0.05). Four of the loci implicated in IDDM potentially overlap with these regions: the putative IDDM6, IDDM9, IDDM13, and DXS998 loci. The first two of these candidate regions, defined in the RA genome scan by the markers D18S68-D18S61-D18S469 (18q22–23) and D3S1267 (3q13), respectively, were studied in 194 additional RA sib pairs from 164 nuclear families. Support for linkage to chromosome 3 only was extended significantly (P = 0.002). The analysis of all 261 families provided a linkage evidence of P = 0.001 and suggested an interaction between this putative RA locus and HLA. This locus could account for 16% of the genetic component of RA. Candidate genes include those coding for CD80 and CD86, molecules involved in antigen-specific T cell recognition. In conclusion, this first genome scan in RA Caucasian families revealed 14 candidate regions, one of which was supported further by the study of a second set of families.

A genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bipolar affective disorder among the Old Order Amish

Bipolar affective disorder (BPAD; manic-depressive illness) is characterized by episodes of mania and/or hypomania interspersed with periods of depression. Compelling evidence supports a significant genetic component in the susceptibility to develop BPAD. To date, however, linkage studies have attempted only to identify chromosomal loci that cause or increase the risk of developing BPAD. To determine whether there could be protective alleles that prevent or reduce the risk of developing BPAD, similar to what is observed in other genetic disorders, we used mental health wellness (absence of any psychiatric disorder) as the phenotype in our genome-wide linkage scan of several large multigeneration Old Order Amish pedigrees exhibiting an extremely high incidence of BPAD. We have found strong evidence for a locus on chromosome 4p at D4S2949 (maximum genehunter-plus nonparametric linkage score = 4.05, P = 5.22 × 10−4; sibpal Pempirical value <3 × 10−5) and suggestive evidence for a locus on chromosome 4q at D4S397 (maximum genehunter-plus nonparametric linkage score = 3.29, P = 2.57 × 10−3; sibpal Pempirical value <1 × 10−3) that are linked to mental health wellness. These findings are consistent with the hypothesis that certain alleles could prevent or modify the clinical manifestations of BPAD and perhaps other related affective disorders.