Genomic screening identifies novel linkages and provides further evidence for a role of MYH9 in nonsyndromic cleft lip and palate.

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth anomaly that requires prolonged multidisciplinary rehabilitation. Although variation in several genes has been identified as contributing to NSCLP, most of the genetic susceptibility loci have yet to be defined. To identify additional contributory genes, a high-throughput genomic scan was performed using the Illumina Linkage IVb Panel platform. We genotyped 6008 SNPs in nine non-Hispanic white NSCLP multiplex families and a single large African-American NSCLP multiplex family. Fourteen chromosomal regions were identified with LOD>1.5, including six regions not previously reported. Analysis of the data from the African-American and non-Hispanic white families revealed two likely chromosomal regions: 8q21.3-24.12 and 22q12.2-12.3 with LOD scores of 2.98 and 2.66, respectively. On the basis of biological function, syndecan 2 (SDC2) and growth differentiation factor 6 (GDF6) in 8q21.3-24.12 and myosin heavy-chain 9, non-muscle (MYH9) in 22q12.2-12.3 were selected as candidate genes. Association analyses from these genes yielded marginally significant P-values for SNPs in SDC2 and GDF6 (0.01

PREDICTION OF DNA METHYLATION BASED ON GENOMIC ARCHITECTURE AND APPLICATIONS OF POSITIONAL WEIGHT MATRICES

Gene silencing due to epigenetic mechanisms shows evidence of significant contributions to cancer development. We hypothesis that the genetic architecture based on retrotransposon elements surrounding the transcription start site, plays an important role in the suppression and promotion of DNA methylation. In our investigation we found a high rate of SINE and LINEs retrotransposon elements near the transcription start site of unmethylated genes when compared to methylated genes. The presence of these elements were positively associated with promoter methylation, contrary to logical expectations, due to the malicious effects of retrotransposon elements which insert themselves randomly into the genome causing possible loss of gene function. In our genome wide analysis of human genes, results suggested that 22% of the genes in cancer were predicted to be methylation-prone; in cancer these genes are generally down-regulated and function in the development process. In summary, our investigation validated our hypothesis and showed that these widespread genomic elements in cancer are highly associated with promoter DNA methylation and may further participate in influencing epigenetic regulation.

Long range physical map of the Wilms' tumor - aniridia region on human chromosome 11

Nephroblastoma or Wilms' tumor is a pediatric renal malignancy that is the most frequently occurring childhood solid tumor. Approximately 1-2% of children with Wilms' tumor also present with aniridia, a congenital absence of all or part of the iris of the eye. These children also have high rates of genitourinary anomalies and mental retardation resulting in what is called the WAGR (Wilms' tumor, aniridia, genitourinary anomaly, mental retardation) syndrome. Cytogenetic analysis of metaphase chromosomes from these patients revealed a consistent deletion of band P13 on chromosome 11. These observations suggest close physical linkage between the disease-related loci, and further imply that development of each phenotype results from the loss of normal gene function.^ The objective of this work is to understand the molecular events at chromosome band 11p13 that are essential to the development of sporadic Wilms' tumor and sporadic aniridia. Two human/hamster somatic cell hybrids have been used to identify sixteen independent DNA probes that map to this segment of the human genome. These newly identified DNA probes and four previously reported probes (CAT, FSHB, D11S16, and HBVIS) have been used to subdivide 11p13 into five intervals defined by overlapping constitutional deletions from several WAGR patients. A long-range physical map of 11p13 has been constructed using each of these probes in Southern blot analysis of genomic DNA after digestion with infrequently cutting restriction enzymes and pulse-field gel electrophoresis. This map, established primarily with MluI and NotI, spans approximately 13 $\times$ 10$\sp{6}$ bp and encompasses deletion and translocation breakpoints associated with genitourinary anomalies, aniridia, and sporadic Wilms' tumor. This complete physical map of human chromosome band 11p13 enables us to localize the genes for sporadic Wilms' tumor and sporadic aniridia to a small number of specific NotI fragments. ^

Analysis of mutations in $\beta$-tubulin genes affecting tubulin stability and assembly

Cmd4 is a colcemid-sensitive CHO cell line that is temperature sensitive for growth and expresses an altered $\beta$-tubulin, $\beta\sb1$. One revertant of this cell line, D2, exhibits a further alteration in $\beta\sb1$ resulting in an acidic shift in its isoelectric point and a decrease in its molecular weight to 40 kD, as measured by two dimensional gel electrophoresis. This $\beta$-tubulin variant has been shown to be assembly-defective and unstable. Characterization of the mutant $\beta\sb1$ in D2 by high pressure liquid chromatography (HPLC) revealed the loss of methionine containing tryptic peptides 7,8,9, and 10. Southern analysis of the genomic DNA digested with several different restriction enzymes resulted in the appearance of new restriction fragments 250 base pairs shorter than the corresponding fragments from the wild-type $\beta\sb1$-tubulin gene. Northern analysis on mRNA from D2 revealed two new message products that also differed by 250 bases from the corresponding wild type $\beta$-tubulin transcripts. To precisely define the region of the alteration, cloning and sequencing of the mutant and wild type genomic $\beta$-tubulin genes were conducted. A size-selected EcoRI genomic library was prepared using the Stratagene lambda Zap II phage cloning system. Using subclones of CHO $\beta$-tubulin cDNA as probes, a 2.5 kb wild type clone and a 2.3 kb mutant clone were identified from this library. Each of these was shown to contain a portion of the gene extending from intron 3 through the end of the coding sequence in exon 4 and into the 3$\sp\prime$ untranslated region on the basis of alignment with the published human $\beta$-tubulin sequence. Sequencing of the mutant 2.3 kb clone revealed that the mutation is due to a 246 base pair internal deletion in exon 4 (base pair 756-1001) that encodes amino acids 253-334. This deletion results in the loss of a putative binding site for GTP which could potentially explain the phenotype of this mutant $\beta$-tubulin. Also sequence comparison of the 3$\sp\prime$ untranslated region between different species revealed the conservation of 200 base pairs with 78% homology. It is proposed that this region could play an important role in the regulation of $\beta$-tubulin gene expression. ^

TUMOR PROGRESSION, METASTATIC POTENTIAL AND GENOMIC INSTABILITY

To investigate the hypothesis that increased malignant potential correlates with increased levels of genetic instability, the following parameters of instability were measured: (1) spontaneous mutation rates for ouabain resistance in murine cell lines of different malignant potentials, (2) the background prevalence of 6-thioguanine (6-TG) resistance in clone 4 (highly metastatic) and clone 19 (poorly metastatic) of the K1735 murine melanoma, (3) the prevalence of ouabain resistant variants in three murine cell lines and their variants after exposure to the mutagen MNNG, (4) the rate of generation of major karyotypic abnormalities in B16 F1 (poorly metastatic) and B16 F10 (highly metastatic) murine melanoma, and (5) analysis of the G-banded karyotypes of cloned B16 F1 and B16 F10 melanoma.^ No correlation of increased spontaneous mutation rates with increased malignant potential was found in repeated experiments with three murine cell lines and their variants of different malignant potential. The background prevalence of g-TG resistance was not significantly different for the poorly and highly metastatic clones of K1735 melanoma. The studies with MNNG-induced mutation showed no increased sensitivity of the highly metastatic variants of the three murine cell lines to mutagenesis. Neither did the rate of generation of major karyotypic abnormalities correlate with malignant potential. However, certain karyotypic differences were demonstrated after G-banding of the B16 F1 and F10 melanomas.^ One hypothesis which is consistent with these results is that the rate of generation of genetic abnormalities need not be strongly related to the degree of malignant potential. An increased prevalence of genetic changes may merely reflect the accumulation of abnormalities while their rate of production remains constant. The presence of specific nonrandom changes likely is the main determinant of malignant potential rather than the rate of production of random changes. ^

A FUNCTIONAL VARIANT OF HLA-DR1 DELINEATED BY T-LYMPHOCYTE CLONE REACTIVITY, PROTEIN CONFORMATION, AND GENOMIC RESTRICTION SITES

This research characterized a serologically indistinguishable form of HLA-DR1 that: (1) cannot stimulate some DR1-restricted or specific T-lymphocyte clones; (2) displays an unusual electrophoretic pattern on two dimensional gels; and (3) is marked by a polymorphic restriction site of the alpha gene. Inefficient stimulation of some DR1-restricted clones was a property of DR1$\sp{+}$ cells that shared HLA-B14 on the same haplotype and/or were carriers of 21-hydroxylase (21-OH) deficiency. Nonclassical 21-OH deficiency frequently demonstrates genetic linkage with HLA-B14;DR1 haplotypes and associates with duplications of C4B and one 21-OH gene. Cells having both stimulatory (DR1$\sb{\rm n}$) and nonstimulatory (DR1$\sb{\rm x}$) parental haplotypes did not mediate proliferation of these clones. However, heterozygous DR1$\sb{\rm x}$, 2 and DR1$\sb{\rm x}$, 7 cells were efficient stimulators of DR2 and DR7 specific clones, respectively, suggesting that a trans acting factor may modify DR1 alleles or products to yield a dominant DR1$\sb{\rm x}$ phenotype. Incompetent stimulator populations did not secrete an intercellular soluble or contact dependent suppressor factor nor did they express interleukin-2 receptors competing for T-cell growth factors. Two dimensional gel analysis of anti-DR immunoprecipitates revealed, in addition to normal DR$\alpha$ and DR$\beta$ chains, a 50kD species from DR1$\sb{\rm x}$ but not from the majority of DR1$\sb{\rm n}$ or non-DR1 cells. The 50kD structure was stable under reducing conditions in SDS and urea, had antigenic homology with DR, and dissociated after boiling into 34kD and 28kD peptide chains apparently identical with DR$\alpha$ and DR$\beta$ as shown by limited digest peptide maps. N-linked glycosylation and sialation of DRgp50 appeared to be unchanged from normal DR$\alpha$ and DR$\beta$. Bg1II digestion and $DR\alpha$ probing of DR1$\sb{\rm x}$ genomic DNA revealed a 4.5kb fragment while DR1$\sb{\rm n}$ DNA yielded 3.8 and 0.76kb fragments; all restriction sites mapped to the 3$\sp\prime$ untranslated region of $DR\alpha$. Collectively, these data suggest that DRgp50 represents a novel combinatorial association between constitutive chains of DR that may interfere with or compete for normal T cell receptor recognition of DR1 as both an alloantigen and restricting element. Furthermore, extensive chromosomal abnormalities previously mapped to the class III region of B14;DR1 haplotypes may extend into the adjacent class II region with consequent intrusion on immune function. ^

THE TRANSLATION PRODUCTS OF MOLONEY MURINE SARCOMA VIRUS 124 GENOMIC RNA

Murine sarcoma viruses constitute a class of replication-defective retroviruses. Cellular transformation may be induced by these viruses in vitro; whereas, fibrosarcomas may result in animals infected with them in vivo (Tooze, 1973; Bishop, 1978). Hybridization studies suggest that murine sarcoma viruses arose by recombination between nondefective murine leukemia virus sequences and certain cellular sequences present in uninfected mouse cells (Hu et al., 1977). A specific gene product, however, has not been implicated in murine sarcoma virus transformation.^ One line of murine sarcoma virus-producing cells, Mo-MuSV-clone 124, (Ball et al., 1973), was studied biochemically because it mainly produces the sarcoma virus as a pseudotype packaged with helper murine leukemia virus proteins. The sarcoma viral RNA was translated in a sophisticated cell-free protein synthesizing system (Murphy and Arlinghaus, 1978). The translation products were analyzed by a number of techniques, including electrophoresis in denaturing gels of SDS polyacrylamide, immunoprecipitation, and peptide mapping. The major products of the total RNA purified from the virus preparation were shown to have molecular weights of about 63,000 (P63('gag)), 42,000 (P42), 40,000 (P40), 38,000 (P38), and 23,000 (P23). The size class of mRNA coding for each of the cell-free products was estimated using a poly(A) selection technique and sucrose gradient fractionation. These analyses were used to localize the coding information related to each of the in vitro synthesized cell-free products within the sarcoma virus genome.^ The major findings of these studies were: (1) the 5' half of the sarcoma viral RNA codes for the 63,000 dalton polypeptide and 42,000 - 38,000 dalton polypeptides derived from the "gag" gene; and (2) the 3' half of the sarcoma viral RNA codes for a 38,000 dalton polypeptide and possibly derived from the cellular acquired sequences. ^

Synthesis and Characterization of Photoaffinity Probes that Target the 5-HT3 Receptor

Abstract: The 5-HT3 receptor is one of several ion channels responsible for the transmission of nerve impulses in the peripheral and central nervous systems. Until now, it has been difficult to characterize transmembrane receptors with classical structural biology approaches like X-ray crystallography. The use of photoaffinity probes is an alternative approach to identify regions in the protein where small molecules bind. To this end, we present two photoaffinity probes based on granisetron, a well known antagonist of the 5-HT3 receptor. These new probes show nanomolar binding affinity for the orthosteric binding site. In addition, we investigated their reactivity using irradiation experiments.

New generation genomic platforms in investigation of complex diseases and BEN.

(Full text is available at http://www.manu.edu.mk/prilozi). New generation genomic platforms enable us to decipher the complex genetic basis of complex diseases and Balkan Endemic Nephropathy (BEN) at a high-throughput basis. They give valuable information about predisposing Single Nucleotide Polymorphisms (SNPs), Copy Number Variations (CNVs) or Loss of Heterozygosity (LOH) (using SNP-array) and about disease-causing mutations along the whole sequence of candidate-genes (using Next Generation Sequencing). This information could be used for screening of individuals in risk families and moving the main medicine stream to the prevention. They also might have an impact on more effective treatment. Here we discuss these genomic platforms and report some applications of SNP-array technology in a case with familial nephrotic syndrome. Key words: complex diseases, genome wide association studies, SNP, genomic arrays, next generation sequ-encing.

Synthesis and testing of photoaffinity probes for the site-selectivechemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Synthesis and testing of photoaffinity probes for the site-selective chemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.[1] It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photolabile building blocks via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photo-labile moieties that show nanomolar binding affinities for the orthosteric binding site. Further on we developed a stable 5-HT3R overexpressing cell line and a purification method to yield the receptor in a high purity. Currently we are investigating crosslinking experiments and subsequent MS – analysis.

Synthesis and testing of photoaffinity probes for the site-selectivechemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Synthesis and testing of photoaffinity probes for the site-selective chemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Genomic cloning and expression of three murine UDP-galactose: beta-N-acetylglucosamine beta1,3-galactosyltransferase genes.

Based on the detection of expressed sequence tags that are similar to known galactosyltransferase sequences, we have isolated three novel UDP-galactose:beta-N-acetylglucosamine beta1, 3-galactosyltransferase (beta3GalT) genes from a mouse genomic library. The three genes, named beta3GalT-I, -II, and -III, encode type II transmembrane proteins of 326, 422, and 331 amino acids, respectively. The three proteins constitute a distinct subfamily as they do not share any sequence identity with other eucaryotic galactosyltransferases. Also, the entire protein-coding region of the three beta3GalT genes was contained in a single exon, which contrasts with the genomic organization of the beta1,4- and alpha1, 3-galactosyltransferase genes. The three beta3GalT genes were mainly expressed in brain tissue. The expression of the full-length murine genes as recombinant baculoviruses in insect cells revealed that the beta3GalT enzymes share the same acceptor specificity for beta-linked GlcNAc, although they differ in their Km for this acceptor and the donor UDP-Gal. The identification of beta3GalT genes emphasizes the structural diversity present in the galactosyltransferase gene family.

Black probes of Schrödinger spacetimes

We consider black probes of Anti-de Sitter and Schrödinger spacetimes embedded in string theory and M-theory and construct perturbatively new black hole geometries. We begin by reviewing black string configurations in Anti-de Sitter dual to finite temperature Wilson loops in the deconfined phase of the gauge theory and generalise the construction to the confined phase. We then consider black strings in thermal Schrödinger, obtained via a null Melvin twist of the extremal D3-brane, and construct three distinct types of black string configurations with spacelike as well as lightlike separated boundary endpoints. One of these configurations interpolates between the Wilson loop operators, with bulk duals defined in Anti-de Sitter and another class of Wilson loop operators, with bulk duals defined in Schrödinger. The case of black membranes with boundary endpoints on the M5-brane dual to Wilson surfaces in the gauge theory is analysed in detail. Four types of black membranes, ending on the null Melvin twist of the extremal M5-brane exhibiting the Schrödinger symmetry group, are then constructed. We highlight the differences between Anti-de Sitter and Schrödinger backgrounds and make some comments on the properties of the corresponding dual gauge theories.