High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones.

Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by in situ hybridization showed a one to one correspondence with those determined by Southern (DNA) blot analysis of hybrid cell lines containing fragments of chromosome 11. Furthermore, by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally. These results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization.

The HLA-A*0207 peptide binding repertoire is limited to a subset of the A*0201 repertoire.

Quantitative A*0207 peptide binding assays have been developed utilizing HLA transfected cells and affinity purified molecules. By using a panel of single substitution analog peptides, it was demonstrated that A*0207 binds peptides with main anchor specificity at position 2 and the C-terminus similar to A*0201. Previous data indicating that A*0207 (but not A*0201) also requires the presence of D or P in position 3 of its peptide ligands was confirmed by the analysis of additional single substituted analogs. Finally, by analyzing the A*0201 and A*0207 binding capacities of panels of unrelated synthetic peptides, it was found that 8/15 (53.3%) A*0201 binders with D or P in position 3 bound A*0207, while only 5/72 (6.9%) A*0201 binders without D or P in position 3 also bound A*0207. Together, these data indicate that although A*0207 may be included amongst A2 supertype alleles, its peptide binding repertoire is largely limited to a subset of that bound by A*0201.

Comparison and contrast of affinity chromatographic determinations of plasma glycated albumin and total glycated plasma protein.

Techniques for affinity measurement of glycated albumin and for glycated total plasma protein have been developed. The two techniques were contrasted. Both techniques are linear over a 100-fold range of sample concentrations. There appears to be a non-specific early glucose binding phase to non-albumin plasma proteins. Although this phase is detected by radioactive incorporation and thiobarbituric acid, it does not interfere with the affinity determination, which does not appear to detect the early binding species. The correlation of glycated albumin levels with glycated hemoglobin levels is much stronger than that of glycated globulin levels with glycated hemoglobin levels. Due to the large contribution of glycated albumin levels to total glycated serum protein levels, the correlation of the latter with glycated hemoglobin levels is sufficiently strong to allow the use of either technique as an adequate index of glycation.

A DNA-based vaccine for the prevention of human cytomegalovirus-associated diseases.

Multiple lines of evidence indicate that in the transplant population human cytomegalovirus (HCMV) infection and its associated diseases are controlled by humoral and cellular immune responses similar to those that arise in asymptomatic, healthy individuals during a naturally-acquired infection. The dominant antibody response to HCMV is to the major surface glycoprotein B (gB) and the dominant cellular immune response is to the tegument phosphoprotein (pp65). We propose that an immunotherapeutic plasmid DNA (pDNA) vaccination approach that induces the requisite responses to major immunological targets of HCMV may provide relief from HCMV-associated diseases in the transplant setting. We have developed gene-based immunotherapeutic products consisting of pDNAs encoding gB and pp65 of HCMV. When tested individually in mice, both pDNAs were highly immunogenic. Relative to vaccination with either gB or pp65 pDNA delivered alone, vaccination with gB and pp65 pDNAs delivered together in phosphate-buffered saline (PBS) elicited reduced antibody and T cell responses to each antigen. Formulating this bivalent vaccine with a poloxamer-based delivery system (VF-P1205-02A), however, significantly increased the antigen-specific immune responses relative to those induced with the bivalent vaccine in PBS, and completely abrogated the decrease in pp65-specific T cell responses observed in mice covaccinated with the pDNAs in PBS. Based on these data, and a favorable safety and toxicity profile in preclinical studies, the bivalent HCMV vaccine consisting of gB and pp65 pDNAs delivered with VF-P1205-02A has advanced to human clinical trials.

Rescuing YAC-insert ends as E. coli plasmids.

End clones from YACs (terminal fragments of the YAC-insert DNA cloned into a plasmid vector) are essential ingredients for contig building using chromosome walking strategies, for fluorescent in situ hybridization experiments, and for generating repeat-free probes for pulsed-field gel electrophoresis or genetic linkage analysis. The basic protocol describes a method for rescuing the CEN (centromere) ends of YACs constructed in the vector pYAC4. However, because this method relies on XhoI and SalI sites, which are relatively rare in the mammalian genome, it is not always possible to obtain a subclone in this manner. An alternate protocol presents a method utilizing integrative plasmid-rescue vectors to facilitate the isolation of both ends of any YAC clone even in the absence of convenient restriction enzyme sites.

Gross bilateral reflux in infants: gradual decrease of initial detrusor hypercontractility.

PURPOSE: We performed followup investigations of the urodynamic patterns of 11 boys with gross bilateral reflux, which are sometimes characterized by low bladder capacity and hypercontractility of the detrusor. MATERIALS AND METHODS: Urodynamic evaluations were done a mean of 2 years after the initial assessment during infancy. RESULTS: Initial hypercontractility resolved and bladder capacity increased from below normal to high in the majority of cases. The number of children with instability did not change significantly. CONCLUSIONS: The urodynamic pattern at followup was similar to that previously reported as the most common dysfunction in older children with reflux.

Immunoglobulin enhancer and promoter motifs 5' of the B29 B-cell-specific gene.

B29 is a B-cell-specific member of the immunoglobulin gene superfamily that is expressed throughout B-cell development beginning with the earliest precursor B cells undergoing immunoglobulin heavy-chain gene segment rearrangements. We have analyzed the region upstream of the B29 gene to identify DNA sequences involved in transcriptional regulation of this gene. The B29 gene lacks a TATA box and transcription is initiated at multiple sites. The B29 gene sequence 5' of these transcription start sites contains six promoter and enhancer motifs known to control immunoglobulin gene transcription. The most notable is a perfect octamer (5'-ATTTGCAT-3'), which binds the Oct-2 B-cell-specific transcription factor and thereby can account for the tissue-specific expression of this gene.

Developmental expression of glial-specific mRNAs in primary cultures of rat brain visualized by in situ hybridization.

The localization of mRNAs which encode the glial-specific marker proteins, glial fibrillary acidic protein (GFAP), glycerol phosphate dehydrogenase (GPDH, EC 1.1.1.8), and myelin basic protein (MBP), was mapped by in situ hybridization in primary cultures of 1-2-day-old rat brain in serum-supplemented medium. Developmental changes of these expressed mRNAs were examined after various times in culture ranging from 8 to 50 days and were correlated with the histological, morphological, and positional characteristics of the cells. By day 8, the culture stratified into a population of flat polygonal astrocytes covered by another population of phase-dark process-bearing cells. When counterstained with May-Grunwald histological stain, astrocytes appeared pale blue, whereas two subpopulations of phase-dark cells stained differentially; one was dark blue while the other was red and smaller. GFAP-specific sequences were abundant at day 8, increased in the astrocyte bedlayer as the culture became confluent, and plateaued at approximately day 16. A minor proportion of blue phase-dark cells contained GFAP mRNA although at a lower abundance. In contrast, GPDH mRNA positive blue phase-dark cells were seen scattered throughout the upper layer of the culture and also around the perimeter of large clumps of red phase-dark cells. These cells were infrequent at day 8 but increased in number at later time points. The expression of MBP mRNA differed from GPDH in that it was more abundant at early time points, plateaued between day 20 and day 24, and was predominantly localized in red phase-dark cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of urodynamic and free voiding pattern in infants with dilating reflux.

PURPOSE: We compared simultaneous investigations of free voiding pattern and urodynamic assessment in infants with dilating reflux to obtain further information on previously suspected bladder dysfunction based on abnormal urodynamic findings. MATERIAL AND METHODS: A total of 33 male and 8 female infants with dilating reflux were included in the study. Free voiding pattern was determined by 4-hour voiding observation compared to previously described voiding pattern studies of healthy infants. Simultaneous invasive urodynamic assessments were performed. RESULTS: The patients were grouped according to urodynamic bladder capacity. Half of the male patients had low bladder capacity with high voiding pressure levels (hypercontractile) and the other half had either normal or high capacity bladders. The low capacity group had frequent small voids and a high rate of interrupted voiding, the high capacity group had infrequent voids of high volumes with high residual urine, and the pattern of the normal capacity group differed only from that of healthy infants by an increase in residual urine. All female infants had the typical characteristics of high capacity bladder on free voiding and urodynamic assessments. CONCLUSIONS: Infants with abnormal invasive urodynamic investigations, including those with a small capacity hypercontractile bladder and those with a high capacity bladder, could be identified on free voiding studies, indicating that an abnormal urodynamic pattern represents bladder dysfunction.

Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast.

Yeast artificial chromosomes (YACs) provide a powerful tool for the isolation and mapping of large regions of mammalian chromosomes. We developed a rapid and efficient method for the isolation of DNA fragments representing the extreme ends of YAC clones by the insertion of a rescue plasmid into the YAC vector by homologous recombination. Two rescue vectors were constructed containing a yeast LYS2 selectable gene, a bacterial origin of replication, an antibiotic resistance gene, a polylinker containing multiple restriction sites, and a fragment homologous to one arm of the pYAC4 vector. The 'end-cloning' procedure involves transformation of the rescue vector into yeast cells carrying a YAC clone, followed by preparation of yeast DNA and transformation into bacterial cells. The resulting plasmids carry end-specific DNA fragments up to 20 kb in length, which are suitable for use as hybridization probes, as templates for direct DNA sequencing, and as probes for mapping by fluorescence in situ hybridization. These vectors are suitable for the rescue of end-clones from any YAC constructed using a pYAC-derived vector. We demonstrate the utility of these plasmids by rescuing YAC-end fragments from a human YAC library.

Molecular localization of the t(11;22)(q24;q12) translocation of Ewing sarcoma by chromosomal in situ suppression hybridization.

Chromosome translocations are associated with a variety of human leukemias, lymphomas, and solid tumors. To localize molecular markers flanking the t(11;22) (q24;q12) breakpoint that occurs in virtually all cases of Ewing sarcoma and peripheral neuroepithelioma, high-resolution chromosomal in situ suppression hybridization was carried out using a panel of cosmid clones localized and ordered on chromosome 11q. The location of the Ewing sarcoma translocation breakpoint was determined relative to the nearest two cosmid markers on 11q, clones 23.2 and 5.8, through the analysis of metaphase chromosome hybridization. By in situ hybridization to interphase nuclei, the approximate physical separation of these two markers was determined. In both Ewing sarcoma and peripheral neuroepithelioma, cosmid clone 5.8 is translocated from chromosome 11q24 to the derivative chromosome 22 and a portion of chromosome 22q12 carrying the leukemia inhibitory factor gene is translocated to the derivative chromosome 11. The physical distance between the flanking cosmid markers on chromosome 11 was determined to be in the range of 1000 kilobases, and genomic analysis using pulsed-field gel electrophoresis showed no abnormalities over a region of 650 kilobases in the vicinity of the leukemia inhibitory factor gene on chromosome 22. This approach localizes the Ewing sarcoma breakpoint to a small region on chromosome 11q24 and provides a rapid and precise technique for the molecular characterization of chromosomal aberrations.

Chromosomal in situ hybridization using yeast artificial chromosomes.

Large DNA fragment cloning methods using yeast artificial chromosomes (YACs) have vastly improved the strategies for constructing physical maps of regions of complex genomes, as well as for isolating and cloning genes important for human disease. We present here a simple and rapid method for carrying out in situ hybridization to metaphase chromosomes using isolated YAC clones by labeling DNA directly in agarose gel slices. Nonisotopic labeling and chromosomal in situ hybridization can be used to determine the chromosomal localization of individual YAC clones on human metaphase chromosomes. This method can also be used to characterize YAC clones consisting of single fragments from those that contain concatamerized, and thus artifactual, inserts. This technique also offers a valuable tool to study consistent translocations in neoplastic diseases by identifying YACs that span a specific chromosomal breakpoint.

Cosmid linking clones localized to the long arm of human chromosome 11.

Molecular probes that contain DNA flanking CpG-rich restriction sites are extremely valuable in the construction of physical maps of chromosomes and in the identification of genes associated with hypomethylated HTF (HpaII tiny fragment) islands. We describe a new approach to the isolation and characterization of linking clones in arrayed chromosome-specific cosmid libraries through the large-scale semiautomated restriction mapping of cosmid clones. We utilized a cosmid library representing human chromosome 11q12-11qter and carried out automated restriction enzyme analysis, followed by regional localization to chromosome 11q using high-resolution in situ suppression hybridization. Using this approach, 165 cosmid linking clones containing one or more NotI, BssHII, SfiI, or SacII sites were identified among 960 chromosome-specific cosmids. Furthermore, this analysis allowed clones containing a single site to be distinguished from those containing clusters of two or more rare sites. This analysis demonstrated that more than 75% of cosmids containing a rare restriction site also contained a second rare restriction site, suggesting a high degree of CpG-rich restriction site clustering. Thirty chromosome 11q-specific cosmids containing rare CpG-rich restriction sites were regionally localized by high-resolution fluorescence in situ suppression hybridization, demonstrating that all of the CpG-rich sites detected by this method were located in bands 11q13 and 11q23. In addition, the distribution of (CA)n repetitive sequences was determined by hybridization of the arrayed cosmid library with oligonucleotide probes, confirming a random distribution of microsatellites among CpG-rich cosmid clones. This set of reagent cosmid clones will be useful for physical linking of large restriction fragments detected by pulsed-field gel electrophoresis and will provide a new and highly efficient approach to the construction of a physical map of human chromosome 11q.

Localization of the human oncostatin M gene (OSM) to chromosome 22q12, distal to the Ewing's sarcoma breakpoint.

Using fluorescence in situ hybridization, a cosmid clone containing the gene for oncostatin M (OSM) was mapped to human chromosome 22q12, placing the OSM gene in the same chromosome band as the leukemia-inhibitory factor gene (LIF). The location of the OSM gene was determined relative to the t(11;22)(q24;q12) of Ewing's sarcoma and found to be distal to the translocation breakpoint on chromosome 22. Analysis of physical distances by pulsed-field gel electrophoresis demonstrated further that the two genes lie within 500 kb of each other.

B29: a member of the immunoglobulin gene superfamily exclusively expressed on beta-lineage cells.

A number of the glycoproteins identified on the surfaces of cells of the immune response belong to the immunoglobulin superfamily. We have isolated and characterized cDNA clones and the complete genomic gene encoding a B-cell-specific member of the immunoglobulin superfamily called "B29." This isolate is expressed at all stages in B-cell development beginning with the earliest precursor B cells undergoing immunoglobulin heavy chain gene diversity region----joining region gene (DH----JH) rearrangements. The protein sequence predicted by the B29 coding region contains a leader sequence and a single extracellular immunoglobulin-like domain, followed by a hydrophobic transmembrane segment and a charged intracytoplasmic domain. The immunoglobulin-like domain contains cysteines and other conserved amino acids characteristic of light chain variable and joining regions, but overall the sequence is only distantly related to immunoglobulins. Each of these domains is encoded in separate exons in the B29 gene, in analogy to other members of the immunoglobulin superfamily. The conserved structural features of the immunoglobulin-like domain in the B29 gene product resemble those of other members of the immunoglobulin superfamily involved in cell recognition and adhesion.

GDP-L-fucose pyrophosphorylase. Purification, cDNA cloning, and properties of the enzyme.

The enzyme that catalyzes the formation of GDP-L-fucose from GTP and beta-L-fucose-1-phosphate (i.e. GDP-beta-L-fucose pyrophosphorylase, GFPP) was purified about 560-fold from the cytosolic fraction of pig kidney. At this stage, there were still a number of protein bands on SDS gels, but only the 61-kDa band became specifically labeled with the photoaffinity substrate, azido-GDP-L-[32P]fucose. Several peptides from this 61-kDa band were sequenced and these sequences were used for cloning the gene. The cDNA clone yielded high levels of GFPP activity when expressed in myeloma cells and in a baculovirus system, demonstrating that the 61-kDa band is the authentic GFPP. The porcine tissue with highest specific activity for GFPP was kidney, with lung, liver, and pancreas being somewhat lower. GFPP was also found in Chinese hamster ovary, but not Madin-Darby canine kidney cells. Northern analysis showed the mRNA in human spleen, prostate, testis, ovary, small intestine, and colon. GFPP was stable at 4 (o)C in buffer containing 50 mM sucrose, with little loss of activity over a 9-day period. GTP was the best nucleoside triphosphate substrate but significant activity was also observed with ITP and to a lesser extent with ATP. The enzyme was reasonably specific for beta-L-fucose-1-P, but could also utilize alpha-D-arabinose-1-P to produce GDP-alpha-D-arabinose. The product of the reaction with GTP and alpha-L-fucose-1-P was characterized as GDP-beta-L-fucose by a variety of chemical and chromatographic methods.

Binding of a peptide antigen to multiple HLA alleles allows definition of an A2-like supertype.

Direct MHC binding assays with radiolabeled peptides and HLA class I-expressing mammalian cells such as EBV-transformed B cell lines and PHA-activated blasts have been developed. Significant binding of the radiolabeled probe could be obtained if the target cells were preincubated overnight at 26 degrees C in the presence of beta 2-microglobulin. Under these conditions, up to a few percent of the HLA molecules expressed by either cell type could be bound by the labeled peptides. With these assays, the degree of cross-reactivity of the A*0201-restricted hepatitis B virus core 18-27 peptide with other A2 subtypes was examined. It was determined that this peptide epitope also binds the A*0202, A*0205, and A*0206 but not A*0207 subtypes. Inhibition experiments with panels of synthetic peptide analogues underlined the similar ligand specificities of the HLA-A*0201, A*0202, and A*0205 alleles. Analysis of the polymorphic residues that help form the B and F pockets of various HLA alleles allowed prediction of binding of the hepatitis B virus core 18-27 epitope to two other HLA alleles (HLA-A*6802 and A*6901). Thus, it appears that a family of at least six different HLA-A molecules may share overlapping ligand specificities (aliphatic residues in position 2 and at the C termini). These results suggest that broadly cross-reactive peptide epitopes can be identified and greatly enhance the prospective feasibility of peptide-based vaccination approaches.

B29 gene products complex with immunoglobulins on B lymphocytes.

B29 is a B-lineage-specific gene predicted from sequence information to be a transmembrane member of the immunoglobulin (Ig) superfamily, with a single extracellular Ig-like domain. Its presumptive cytoplasmic region contains a peptide motif present in CD3 and other molecules involved in lymphocyte activation. Affinity-purified goat antibodies were prepared to a TrpE fusion protein of B29 and used to study B29 expression on lymphoid cells. The antiserum precipitated surface-labeled heterodimers from B lymphoma cells. One was 65-88 kDa (unreduced) or 36-47 plus 32-34 kDa (reduced) by SDS/PAGE analysis, regardless of detergent. A smaller heterodimer was detected only with Triton detergent extraction. IgM molecules were coprecipitated by the B29 antiserum when the weak detergent digitonin was used. In addition, cocapping experiments revealed that most B29 molecules codistribute with Ig on the cell surface. Although early B-lineage cells and plasma cells contain B29 mRNA, surface expression was detectable only on B cells that had significant amounts of surface Ig. The surface expression was B-lineage-specific and included cells from mutant xid mice and B-cell lines representing mu, delta, gamma, and alpha heavy-chain isotypes and both kappa and lambda light-chain types. The density of surface B29 protein correlated directly with surface mu heavy-chain density on subclones of a B-cell lymphoma and lipopolysaccharide-stimulated pre-B cells. These findings show that B29 is covalently linked in a heterodimer and are consistent with a recently proposed model of surface Ig complexes.