Chromosomes in solid tumors and beyond.
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Chromosome Aberrations↗
Biomedical subjects
R M Gemmill
Publications and source records attributed to R M Gemmill.
Deletion mapping of the beta-glucuronidase gene.
GUSB, the gene for beta-glucuronidase, has been localized to the proximal long arm of chromosome 7 between 7q11.2 and 7q22. Deficiency of beta-glucuronidase results in mucopolysaccharidosis type VII (MPS VII, Sly syndrome). The enzymatic defect has been demonstrated in cultured skin fibroblasts, leukocytes and serum of affected patients. An 8-yr-old boy presented with manifestations similar to MPS VII (mental retardation, short stature, "coarse" facial appearance, mild skeletal involvement and recurrent lower respiratory tract infection) but other, discrepant abnormalities, e.g., bilateral iris colobomata and cleft palate. Normal activity of beta-glucuronidase was found in the patient's leukocytes. Chromosome analysis disclosed an interstitial deletion of 7q with one breakpoint at the interface between bands 11.22 and 11.23 and the other breakpoint within band 21.1. DNA from this patient's leukocytes was analyzed for dosage of GUSB sequences. This locus appeared to be present at the normal diploid level. These findings suggest that GUSB is not in the portion of chromosome 7 deleted in our case, narrowing the smallest region of overlap to 7q21.1----7q22. We therefore assign the beta-glucuronidase gene to 7q21.1----7q22.
Child↗
Translocation t(3;8)(p14.2;q24.1) in renal cell carcinoma affects expression of the common fragile site at 3p14(FRA3B) in lymphocytes.
The common fragile site at 3p14(FRA3B) is cytogenetically close to the positions of translocation and deletion breakpoints frequently observed in renal cell carcinoma (RCC) and small cell carcinoma of the lung. Possible involvement of this fragile site in the familial RCC t(3;8)(p14.2;q24.1) was investigated. Expression of FRA3B, induced by treatment of lymphocytes with aphidicolin, is altered by the translocation. These results suggest that the fragile site is very close to, if not coincident with, the translocation breakpoint.
Aphidicolin↗
Loss of common 3p14 fragile site expression in renal cell carcinoma with deletion breakpoint at 3p14.
The common fragile site in human chromosome band 3p14 is a constant cytogenetic marker present on every normal chromosome #3. Therefore, we selected a renal cell carcinoma with a deletion breakpoint in 3p14 for analysis of the 3p14 fragile site. Aphidicolin was used to induce the expression of the 3p14 fragile site. The fragile sites expressed in the renal carcinoma cells generally mirrored those expressed in lymphocytes. The normal chromosome #3 in the renal carcinoma cells expressed the common 3p14 fragile site. The partially deleted #3 did not. The deletion breakpoint, therefore, cannot be beyond the 3p14 fragile site. The common fragile site in 3p14 must be at or very near the deletion breakpoint in 3p14 in renal cell carcinoma. These results are consistent with this fragile site causing this cancer chromosome deletion.
Aphidicolin↗
Structural organization of the alpha-amylase gene locus in Drosophila melanogaster and Drosophila miranda.
Chromosomal sites belonging to the alpha-amylase gene family have been identified in D. melanogaster and D. miranda and in the sibling species of miranda, pseudoobscura, and persimilis. Two sites occur in chromosome 2 of melanogaster; one contains the Amy gene locus (54A) and the other an amylase "pseudogene" (53CD). Two sites of homology exist at 73A and 78C and perhaps another at 81BC in chromosome 3 of pseudoobscura and persimilis and in the homologous regions of the X2 chromosome in miranda. The active Amy locus is apparently at 73A. The structural organization of cloned sequences from this multigene family in melanogaster and miranda is under analysis, with emphasis on the functional Amy gene region. Electrophoretic variants of amylase have served as invaluable tools in these studies. For melanogaster, their use as genetic markers enabled us to positively identify our lambda Dm65 clone of the Amy locus and to show that it contains two functional copies of the structural gene for alpha-amylase. Amylase isozymes are now being used in P element-mediated transformation experiments aimed at defining regulatory elements for the temporal and spatial control of amylase expression during development and in response to dietary glucose. In miranda, electrophoretic variants of amylase were useful in assigning the Amy locus to chromosome X2, and they continue to serve as essential markers in our study of the evolution of dosage compensation for amylase expression in males of this species. Restriction maps of the Amy locus in 7 strains of D. melanogaster indicate that despite the worldwide origins of the chromosome samples, all contain a duplication of the amylase structural gene at this locus regardless of whether they produce two alpha-amylase isozymes, a single variant, or none. We have aligned these maps with the genetic and cytological maps of chromosome 2R in melanogaster and assigned alleles for different amylase isozymes to either the proximal or distal Amy gene copy in a number of strains. Restriction site polymorphism is relatively limited at the Amy locus, but some strain-specific rearrangements exist. The locus of two strains with reduced amylase activity, Amy1 (CA 1) and Amy "null", contain anomalies--an insertion in the former and an inversion in the latter. Causal relationships are being sought between the level of amylase expression in these strains and the position of their respective anomalies.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Y chromosome--specific DNA sequences in Turner-syndrome mosaicism.
Phenotypic females with Y-chromosomal material in their genome have an increased risk for development of gonadal malignancy. The detection and identification of Y-chromosomal material in these cases can be of critical importance for medical management. Chromosome analysis in four patients with Turner syndrome revealed the characteristic 45,X chromosome complement together with a second cell population containing a small marker chromosome (46,X, + mar). Molecular-hybridization analyses utilizing cloned, Y chromosome-specific DNA sequences were performed to determine whether Y-chromosomal material was present in each patient. Three cases contained some Y chromosome-specific sequences, whereas one case was negative with all four probes that we used. These results were compared with detailed cytogenetic studies--including G-, Q-, and G-11-banding--of the marker chromosomes. In one case in which Y chromosome-specific DNA sequences were demonstrated, the marker chromosome was G-11 negative. These results demonstrate that cytogenetic analysis alone can lead to misidentification of some Y chromosome-derived markers. The combination of cytogenetic and molecular analyses permits a more accurate characterization of anomalous Y chromosomes and in turn provides additional information that can be crucial to the correct medical management of Turner-syndrome patients.
Chromosome Banding↗
Separation of large DNA molecules by modified pulsed field gradient gel electrophoresis.
Resolution of DNA fragments by pulsed field gradient gel electrophoresis is a function of the pulse time, geometry, and strength of the orthogonal electric fields. The first field geometry described had a number of disadvantages. We show that these disadvantages can be largely overcome by a modified electric field geometry together with an altered switch pattern. These changes are shown to have critical consequences for the technique. Resolution is more uniform across the gel, which permits more samples to be analyzed on the same gel. In addition, DNA molecules follow a migration path that is approximately straight down the gel. This aspect also increases the number of usable wells. One important property of the system described here provides some insight into the mechanism whereby DNA molecules are resolved by this method.
DNA↗
Structural organization of the Amy locus in seven strains of Drosophila melanogaster.
Restriction maps were made by Southern blot analysis of the Amy (alpha-amylase) region in 7 strains of D. melanogaster using endonucleases SalI, XhoI and EcoRI. These were compared to the map of lambda Dm65 which contains the cloned Amy region. Strains used produce either two amylase variants, a single variant, or no amylase, yet all 7 strains carry two Amy genes as inverted repeats at the Amy locus. This and the orientation of the repeats resembles the situation in lambda Dm65. Most restriction sites mapped are conserved but two strains contain a large insertion which differs in size and position between strains. A complex anomaly, probably an inversion, exists at the Amy locus in a null strain. Maps for our Amy1,3 strain and the lambda Dm65 clone are identical, the DNA of each having been derived from a Canton-S wild stock. Restriction and genetic maps of the Amy region were aligned and alleles assigned to the proximal and distal genes, Amy-p and Amy-d.
Animals↗
The Philadelphia chromosome: a model of cancer and molecular cytogenetics.
Recent developments in molecular biology related to the Ph chromosome lead us to an evaluation of knowledge regarding this chromosome. The molecular advances are related to two cellular oncogenes, c-abl and c-sis, and also to the identification and molecular cloning of specific areas of DNA (e.g., band 22q11), permitting the isolation of a probe specific for the translocation breakpoint domain. In the preponderant number of cases examined, it was found that the breakpoints at 22q11 occur within a limited region of up to 5-6 kb, for which the term "breakpoint cluster region" (bcr) has been suggested. In contrast, breaks at 9q34 seem to occur within a much larger region at the molecular level. Yet to be established is the exact genetic composition of the bcr and a determination as to whether or not the breaks leading to the disease occur preferentially within specific areas. In spite of this level of knowledge, we do not understand how the Ph chromosome participates in CML. If Ph-positive CML is ultimately associated with a cascade of gene activations, the unraveling of their nature and chronology will undoubtedly tell us much of their contribution to the biology of CML, in particular, and to neoplasia, in general. In this respect, the rather clear description of CML in cytogenetic, clinical, and laboratory terms, the relatively long chronic phase of the disease, and the association of the blastic phase with nonrandom chromosome changes (at least in the initial phases of the disease) make Ph-positive CML an excellent candidate for a model for the study of molecular events in human neoplasia.
Chromosome Banding↗
High A + T content conserved in DNA sequences upstream of leuABCD in Escherichia coli and Salmonella typhimurium.
The nucleotide sequence of over 800 base pairs of DNA upstream of leuP was determined for Escherichia coli and Salmonella typhimurium. In both of these enteric bacteria, approximately 500 base pairs of A + T-rich sequences separates leuP from an upstream open reading frame. Although these A + T-rich sequences share little homology, the distribution of A + T base pairs within the region is strikingly conserved. Deletion of the A + T-rich sequences upstream of the E. coli leu operon does not markedly affect the strength of the leu promoter in vivo.
Adenine↗
DNA demethylation induced by 5-azacytidine does not affect fragile X expression.
Experiments were performed to determine the role of DNA demethylation in fragile X expression. Fragile X positive lymphoblastoid cells were treated with 5-azacytidine and harvested for analysis of fragile X expression both directly following treatment and after a recovery period in the absence of the drug. The effectiveness of 5-azacytidine treatment in inducing DNA demethylation was concurrently monitored by analysis of methylation changes at random autosomal loci in isolated DNA from treated cells. Under conditions where 5-azacytidine was found to inhibit fragile X expression, no DNA demethylation was observed. At the time when demethylation did occur, fragile X expression was not affected. These results strongly indicate that DNA demethylation is not involved in fragile X expression.
Azacitidine↗
Translocations in T-cell leukemia and lymphoma.
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Cell Line↗
Molecular cloning of alpha-amylase genes from Drosophila melanogaster. I. Clone isolation by use of a mouse probe.
A cloned alpha-amylase cDNA sequence from the mouse is homologous to a small set of DNA sequences from Drosophila melanogaster under appropriate conditions of hybridization. A number of recombinant lambda phage that carry homologous Drosophila genomic DNA sequences were isolated using the mouse clone as a hybridization probe. Putative amylase clones hybridized in situ to one or the other of two distinct sites in polytene chromosome 2R and were assigned to one of two classes, A and B. Clone lambda Dm32, representing class A, hybridizes within chromosome section 53CD. Clone lambda Dm65 of class B hybridizes within section 54A1-B1. Clone lambda Dm65 is homologous to a 1450- to 1500-nucleotide RNA species, which is sufficiently long to code for alpha-amylase. No RNA homologous to lambda Dm32 was detected. We suggest that the class B clone, lambda Dm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.
Animals↗
Molecular cloning of alpha-amylase genes from Drosophila melanogaster. II. Clone organization and verification.
Restriction maps of an alpha-amylase structural gene clone, lambda Dm65, and of four putative alpha-amylase pseudogene clones are presented. Two alpha-amylase structural genes, inverted with respect to each other, are contained in lambda Dm65. Subregions of internal DNA sequence homology within lambda Dm65 and of cross-homology between the presumptive pseudogene clones and lambda Dm65 were determined. Subregions of cross-homology between the Drosophila clones and the mouse alpha-amylase cDNA clone, pMSa104, were also determined. The presence of functional alpha-amylase structural genes in lambda Dm65 was verified by injection of appropriate subclones into the germinal vesicle of Xenopus oocytes, followed by incubation of the oocytes under conditions that allowed coupled transcription and translation of injected genes to occur. Subclones of the 3.8- and 5.6-kb EcoRI fragments of lambda Dm65 were shown to code for alpha-amylase isozymes 1 and 3, respectively, of Drosophila melanogaster Canton-S. Both subclones are homologous to RNA of a size sufficient to accommodate the alpha-amylase-coding information. No RNA species homologous to other subcloned EcoRI fragments of lambda Dm65 was detected.
Animals↗
Promoter mutation causing catabolite repression of the Salmonella typhimurium leucine operon.
Two mutations that affect expression of the Salmonella typhimurium leu operon were investigated. leu operon DNA from these mutant strains was cloned, and nucleotide sequences of the leu control regions were determined. leu-500, which eliminates expression of all four leu genes simultaneously, is a point mutation in the -10 region of the leu promoter. leu-2012 is a point mutation within the -35 region of the leu promoter. leu-2012 suppressed leucine auxotrophy caused by leu-500 only when the medium contained a carbon source that does not cause catabolite repression. A cya mutation (adenylate cyclase deficiency) introduced into the leu-500 leu-2012 strain caused leu enzymes to be made only if cAMP was supplied exogenously. A leu-500 leu-2012 strain containing a crp mutation (cAMP receptor protein deficiency), on the other hand, could not make leu enzymes even in the presence of cAMP. In vitro transcription experiments demonstrated that the leu-2012 mutation created a new transcription initiation site. RNA polymerase utilized this site in vitro in the absence of added cAMP receptor protein and cAMP.
Base Sequence↗
Transcription initiation sites of the leucine operons of Salmonella typhimurium and Escherichia coli.
Evidence for a transcription attenuation site downstream from the leu promoter was obtained by transcription experiments in vitro. Most transcription initiated in vitro from leuP is terminated prematurely, resulting in the synthesis of a 160 nucleotide leader RNA. We define here the point at which transcription is initiated in vitro and in vivo and demonstrate that the site of premature termination is between the promoter and the first structural gene (leuA). Additional nucleotide sequences are presented that extend the known sequence 200 base-pairs upstream and 300 base-pairs downstream from leuP. The location of the promoter-proximal end of cistron leuA was deduced by comparing nucleotide sequence data with the sequence of the ten amino acids at the N-terminus of alpha-isopropylmalate synthase. To facilitate the isolation of quantities of material for sequencing experiments, the enzyme was isolated from a plasmid-containing strain, CV605, grown under conditions of leucine limitation. Under such conditions, about 20% of the total soluble protein of strain CV605 is alpha-isopropylmalate synthase and another 20% is beta-isopropylmalate dehydrogenase (leuB product).
Amino Acid Sequence↗
Genetic mechanism for tissue-specific control of alpha-amylase expression in Drosophila melanogaster.
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Amino Acid Sequence↗
leu operon of Salmonella typhimurium is controlled by an attenuation mechanism.
The nucleotide sequence of the control region of the leu operon of Salmonella typhimurium was determined. A prominent feature of this region is a signal for termination of transcription. In vitro, transcription does terminate at this site, yielding a leader RNA of about 160 nucleotides as a major product. This leader RNA is potentially translatable into a peptide containing 28 amino acids, 4 of which are adjacent leucine residues. Several regions of base complementarity exist within the leader, positioned such that pairing of one region precludes pairing of another. The position of the four leucine codons relative to two regions of base complementarity suggest a model for the regulation of the leu operon similar to that proposed by Yanofsky and coworkers for the trp operon. In addition, a third region of base complementarity was identified which, when incorporated into the model, explains why premature termination is the usual outcome when transcription is initiated in vitro by purified RNA polymerase.
Base Sequence↗