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Biomedical subjects

B D Young

Publications and source records attributed to B D Young.

At least 19 recordsLinked to original sources
PubMed1992

Direct sequence analysis of the t(14;18) chromosomal translocation in Hodgkin's disease.

There have been conflicting reports about the occurrence of the t(14;18) chromosomal translocation in Hodgkin's disease. A polymerase chain reaction analysis of biopsy specimens from 21 patients with Hodgkin's disease (HD) has shown the presence of the t(14;18) translocation in four cases (20%). All four patients had nodular sclerosing HD. Direct sequencing of the amplified 14q+ junctions established that the BCL-2 (major breakpoint region) sequence was fused to an Ig joining region (JH) (J6 in all four cases). Different breakpoints were observed in each case but were similar in nature to the breakpoints described in follicular lymphoma. The exact nature and cell of origin in HD remains obscure, although the presence of the t(14;18) translocation may reflect either a B-cell origin in these cases or associated lymphoid hyperplasia.

Adolescent↗
PubMed1992

Molecular cloning and analysis of chromosome band 11q23 involved in leukaemia-associated translocations.

Three overlapping yeast artificial chromosomes (YACs) spanning a 780 kb region of DNA around the CD3 locus on chromosome 11 have been isolated and characterised. The individual cloned regions have been mapped by in situ hybridisation to chromosome band 11q23, and a restriction enzyme map of this region has been constructed. The positions of these clones in relation to a series of leukaemia-associated chromosomal translocations has also been determined. It was concluded that, although two clones lay entirely proximal to the breakpoints examined, the third clone (13HH4) encompassed the breakpoints for the translocations t(4;11), t(6;11), and t(9;11). The t(9;11) was observed in an acute myeloid leukaemia in a patient previously treated for an unrelated malignancy. It would thus appear that the breakpoints at chromosome band 11q23 occurring in therapy-related leukaemias are in the same region as those found in adult and childhood acute leukaemias and may result from a common underlying mechanism.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Fluorescent in situ identification of human marker chromosomes using flow sorting and Alu element-mediated PCR.

A novel approach to the identification of human chromosomes has been developed. Chromosomal in situ hybridization (or "chromosome painting") has been performed using Alu element-mediated PCR products from small quantities (250-500) of flow-sorted normal and abnormal chromosomes. Chromosome paints for various normal chromosomes, including 5, 6, 7, 14, 18, 19, 21, and 22, were generated and shown to be effective in the identification of the appropriate chromosomes. In addition, certain abnormal chromosomes, including a mental retardation-associated deletion chromosome 11 (q22-q23), the products of the constitutional translocation t(11;22), and the CML-associated t(9;22), were used to generate region-specific paints. In each case, the appropriate regions of the chromosomes were highlighted and this strategy is, therefore, well suited to the identification of previously unidentified marker chromosomes. A further direct consequence of this work is that chromosome paints specific for the common aberrant chromosomes, such as the Philadelphia chromosome, can be generated and made widely available. These may find particular use in the analysis of complex or masked chromosomal translocations.

Base Sequence↗
PubMed1992

A trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias.

Some acute lymphocytic leukaemias, particularly those in young children, are associated with a t(4;11)(q21;q23) reciprocal translocation. We have cloned the translocation breakpoint on chromosome 11q23 and isolated corresponding RNA transcripts from this region. The translocation occurs within a cluster of Alu repetitive elements located within an intron of a gene that gives rise to 11.5 (kb) transcript spanning the translocation breakpoint. The 11.5 kb transcript encodes a protein that is highly homologous to the Drosophila trithorax gene, a developmental regulator. An analysis of a series of leukaemic patients carrying t(4;11) and t(9;11) translocations indicate that the majority of breakpoints in infant leukaemias lie within a 5 kb region.

Amino Acid Sequence↗
PubMed1991

Construction, arraying, and high-density screening of large insert libraries of human chromosomes X and 21: their potential use as reference libraries.

We have constructed cosmid libraries from flow-sorted human chromosomes X and 21, each of which contains greater than 30 genome equivalents, and have developed systems allowing permanent storage of primary clones, easy screening of libraries in high-density filter formats, and the simultaneous generation of fingerprinting and mapping data on the same set of cosmid clones. Clones are picked into microtiter plate wells and stored at -70 degrees C. A semiautomatic robot system allows the generation of filter replicas containing up to 10,000 clones per membrane. Sets of membranes containing 15-20 chromosome equivalents of both chromosomes will be used for the construction of ordered clone libraries by hybridization fingerprinting protocols. In addition, multiple sets of two membranes containing 4 chromosome equivalents of the human X chromosome, and one membrane containing 3 chromosome equivalents of chromosome 21, have been distributed to other interested laboratories as part of a system of reference libraries. This system allows other groups easy access to the clones and offers an efficient protocol to combine results generated in different laboratories using these libraries. Here we describe the construction of the libraries and demonstrate the use of high-density screening filters in oligonucleotide probe hybridizations and the isolation of cosmids by hybridization with probes from the X chromosome.

Chromosomes, Human, Pair 21↗
PubMed1991

Molecular cloning of DNA from a sorted human minichromosome.

A human supernumerary minichromosome (MC), found in a newborn baby and sorted on a fluorescence-activated cell sorter (FACS-440) has been previously described [Ferretti et al., Cytotechnology 1 (1987) 7-12]. We report here on the construction of a library of EcoRI fragments in the phage lambda gtWES.lambda B', starting from 7.5 ng of MC DNA, and describe the isolation of single-copy DNA clones from the library in a two-step procedure. We employed in situ hybridization to unambiguously select the clones specific for the MC, and used three of these clones to demonstrate that it originated from chromosome 9.

Chromosomes, Human, Pair 9↗
PubMed1991

Gene mapping by microdissection and enzymatic amplification: heterogeneity in leukaemia associated breakpoints on chromosome 11.

A new strategy for mapping chromosome translocation breakpoints in relation to known genes has been developed. This approach is based on the amplification by the polymerase chain reaction (PCR) of specific target sequences from small numbers of microdissected chromosome fragments. This method has been applied to leukaemia-associated translocations affecting the q23 region of chromosome 11. In two independent leukaemias, the t(6;11) translocation was distinguished from the t(9;11) and t(4;11) translocations by demonstrating that the former breakpoint on chromosome 11 lay proximal to the CD3D gene while the latter breakpoints lay distal to CD3D. All three translocation breakpoints were found to lie proximal to ETSI and THYI. The data suggest that although these leukaemia-associated breakpoints on chromosome 11 are cytogenetically identical they may involve disruption of different genes. This approach offers a rapid alternative to mapping by hybridisation of probes either in situ to chromosomes or to somatic cell hybrids containing the appropriate derivative chromosomes.

Base Sequence↗
PubMed1991

CD3G is within 200 kb of the leukemic t(4;11) translocation breakpoint.

The t(4;11)(q21;q23) has been associated with acute lymphocytic leukemia (ALL) especially in infants. The t(4;11) breakpoint on chromosome 11 is cytogenetically indistinguishable from breakpoints for other leukemia-associated translocations affecting 11q23. The molecular basis of the t(4;11) is unknown although a number of genes have been mapped to 11q23. The CD3D, G, and E genes have been positioned proximal to the 11q23 breakpoint of the 4;11 translocation while the THY1 and ETS1 genes have been mapped distal to this breakpoint. We report evidence that CD3G is within 200 kb of the 4;11 breakpoint as observed by pulsed field gel analysis. A rearrangement of the CD3G gene has been observed in a cell line derived from a patient with the t(4;11) translocation and in a hybrid cell line containing the derivative 11q chromosome derived from this cell line, using the restriction enzymes SacII and ClaI. Similar rearrangements using SacII were observed in 2 further patients with ALL and the t(4;11) translocation. No rearrangements in the same DNA were observed using ETS1, THY1, and D11S29 and a range of rare cutter restriction enzymes. CD3G thus provides a tool for the cloning and analysis of the 4;11 translocation, and poses a question of its possible involvement at long range with this translocation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Direct sequence analysis of 14q+ and 18q- chromosome junctions at the MBR and MCR revealing clustering within the MBR in follicular lymphoma.

The t(14;18) translocation is a highly consistent feature of follicular lymphoma although the underlying mechanism generating this fusion remains uncertain. The breakpoints on chromosome 18 are at one of two sites, designated mbr and mcr, in the bcl-2 gene. A polymerase chain reaction strategy has been developed for amplification and direct sequencing of the resultant 14q+ and 18q- reciprocal junctions. Sequence analysis of the amplified 14q+ junction established that 21 tumours contained a bcl-2 (mbr) sequence to an immunoglobulin JH region, the majority being J5 or J6. A nonrandom pattern of breakpoints within the mbr region was found. Clustering of the breakpoint occurred with over 60% of the translocations clustering within 10 bases. There was a second cluster within the mbr 50 bases 3' of the first cluster. One of these junctions had an unusual configuration with the bcl-2 and JH sequences separated by a recognisable DH region. This suggests that at least some of the junctional sequences, previously thought of as N insertions, may be fragments of unrecognised DH regions. In one of these tumours it was possible to sequence the reciprocal 18q- junction, showing it to consist of a DH/bcl-2 (mbr) fusion. Analysis of both reciprocal junctions for a translocation in the mcr region of bcl-2, showed that this 18q- junction also consisted of DH fused to a bcl-2 sequence. In contrast to previous analyses, which demonstrated either loss or duplication of bcl-2 sequences at the breakpoints, the bcl-2 sequence was conserved during the mbr and mcr translocations in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

DNA sequences of chromosome 21-specific YAC detect the t(8;21) breakpoint of acute myelogenous leukemia.

The t(8;21)(q22;q22) is a nonrandom translocation specifically marking blasts of acute myelogenous leukemia (AML) with undifferentiated phenotype. The breakpoint on chromosome 21 involved by this rearrangement has been precisely localized relative to cloned DNA markers by physical and genetic linkage analysis enabling the use of positional cloning for its isolation. Yeast artificial chromosome (YAC) clones for loci proximal (D21S65) and distal (ERG) to the (21q22) breakpoint have been developed and their chromosome 21 origin and location relative to the breakpoint has been established. By using in situ hybridization analysis, a 240 kb YAC clone for the D21S65 locus clearly identified both derivative chromosomes of the (8;21) translocation in metaphase spreads of leukemia blasts with the rearrangement. The characterization of the DNA sequences contained in this 240 kb YAC can reveal the functional consequences of their derangement in leukemia with abnormalities of the (21q22) region.

Base Sequence↗
PubMed1991

The generation of DNA probes to chromosome 11q23 by Alu PCR on small numbers of flow-sorted 22q- derivative chromosomes.

A strategy for the isolation of DNA probes from small numbers of flow-sorted human chromosomes has been developed. A lymphoblastoid cell line carrying the 22q- derivative chromosome product of the constitutional t(11;22) translocation was used as the source of chromosomes. Synthetic oligonucleotide primers, based on the consensus Alu sequence, were used to amplify inter-Alu sequence from 500 flow-sorted 22q- derivative chromosomes. The amplified sequences were cloned into a plasmid vector by blunt-end ligation, yielding clones with inserts in the range of 400 to 1000 bp. Approximately 70% of these clones hybridized to human DNA as single-copy probes. To identify clones derived from chromosome 11, the library was screened with a heterogeneous probe prepared by Alu-PCR amplification from the DNA of a somatic cell hybrid containing one homology of chromosome 11. All the positive clones found were mapped to within the q23-q25 region of chromosome 11 known to be translocated onto the 22q- derivative chromosome. Further mapping studies showed that most of these probes (7/8) lay between the breakpoints for the t(4;11) translocation of acute lymphocytic leukemia and the t(11;22) of Ewing sarcoma. Thus, the use of Alu-PCR on the small derivative chromosome 22q- has provided a greatly enriched source of probes to region 11q23, a part of the genome that is currently of great interest. This approach will be particularly appropriate to small numbers of chromosomes when high specificity rather than total representation is required.

Animals↗
PubMed1991

The origin of a morphologically unidentifiable human supernumerary minichromosome traced through sorting, molecular cloning, and in situ hybridisation.

A supernumerary minichromosome has been detected in a severely malformed patient. Attempts at identifying the marker by conventional approaches were unsuccessful. The physical isolation of the minichromosome by fluorescence activated sorting, molecular cloning of its DNA, and in situ hybridisation experiments performed with single copy DNA probes allowed us to show that it was derived from a rearrangement involving the centromere and the proximal region of the short arm of chromosome 9.

Abnormalities, Multiple↗
PubMed1991

The significance of circulating cells carrying t(14;18) in long remission from follicular lymphoma.

Peripheral blood mononuclear cell fractions from 15 patients in continuous clinical remission from follicular lymphoma for longer than 10 years were examined for cells carrying the t(14;18) translocation using the polymerase chain reaction (PCR). The assay used was able to detect one positive cell in approximately 5 x 10(5) cells (a single 14q+ molecule in 2.5 micrograms DNA). Cells positive for t(14;18) were found in six of eight patients initially presenting with stage III or IV disease, compared with zero of seven of those with stage I or II disease (P less than .05). In two cases 14q+ junction regions were also successfully amplified from formalin-fixed biopsy material obtained at presentation 12 and 17 years previously. In both, sequence analysis demonstrated that the cells circulating in remission belonged to the original clone. These results indicate that cells bearing t(14;18) frequently persist in the peripheral blood in long remission of advanced follicular lymphoma and question the value of their presence as a predictor of relapse.

Adult↗
PubMed1991

Bcl-2/JH rearrangements in benign lymphoid tissues with follicular hyperplasia.

The t(14; 18)(q32;q21) chromosomal translocation, characteristic of follicular lymphoma, couples the bcl-2 protooncogene on chromosome 18 to the immunoglobulin heavy-chain joining region (JH). This results in a deregulated transcription rate of bcl-2, suggesting a major role of the t(14;18) translocation in lymphomagenesis. By using a sensitive polymerase chain reaction technique specific for the major breakpoint region t(14;18), we now demonstrate the presence of bcl-2/JH rearrangements in lymph nodes and tonsils with follicular hyperplasia in 13 of 24 cases (54%). The approximate frequency was one translocation-positive cell in 10(5) cells. No bcl-2/JH rearrangements were detected in reactive lymph nodes without follicular hyperplasia or in bone marrow cells. Sequence analysis showed the amplified bcl-2/JH fragments to be unique to each individual sample and distinct from 24 sequenced follicular lymphoma-derived t(14;18) junctions, thus excluding contamination artifacts. The presence of random nucleotide insertions at the breakpoint junctions suggests a pre-B-cell origin of the t(14;18) translocation, in analogy with follicular lymphomas. We conclude that the t(14;18) translocation can occur in non-malignant tissue and will not, on its own, lead to malignancy.

Base Sequence↗
PubMed1991

Establishment of a lymphoblastoid cell line, SD-1, expressing the p190 bcr-abl chimaeric protein.

A lymphoblastoid cell line (SD-1) has been established by Epstein-Barr virus immortalisation of Philadelphia chromosome positive acute lymphoblastic leukaemia (ALL) cells. Using newly derived anti-bcr monoclonal and anti-abl polyclonal antibodies it was demonstrated that both the original leukaemic cells and the derived cell line expressed the p190 form of the bcr-abl protein found in a proportion of cases of Philadelphia chromosome positive ALL. Interestingly, the leukaemia and the derived cell line each displayed different, clonal patterns of immunoglobulin gene rearrangements providing direct evidence that the t(9;22) translocation which results in the expression of the p190 bcr-abl protein must occur before immunoglobulin heavy chain gene rearrangement. In contrast to the leukaemia, which had multiple chromosome abnormalities in addition to the t(9;22), the cell line had the t(9;22) translocation as its sole abnormality. Although SD-1 cells were demonstrated to express continuously the p190 bcr-abl protein, they were unable to form colonies in soft agar and did not cause tumours in splenectomised nude mice. This cell line therefore represents an appropriate target cell line in which to examine the cooperativity of the p190 bcr-abl protein with other activated oncogene products.

Animals↗
PubMed1991

Normal c-abl gene protein--a nuclear component.

The subcellular distribution of the c-abl and bcr-abl gene products from KG1A and K562 cells has been studied by two different techniques. Firstly, physical disruption followed by subcellular fractionation was used to demonstrate that normal c-abl (p145) was recovered from the cytosol and the nuclear fractions of KG1A cells. In contrast, bcr-abl products were recovered exclusively from the cytosol fraction of K562 cells. Secondly, indirect immunofluorescence was used to localize c-abl protein to the cytoplasm, nuclear membrane and infrequently to the nucleus of KG1A cells and bcr-abl protein to only the cytoplasm of K562 cells. Thus both the approaches indicate that there is a component of normal c-abl products which appears to be nuclear and this is not reflected in the distribution of the bcr-abl 210 kDa protein, which remains cytosolic.

Animals↗