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Biomedical subjects

A Bank

Publications and source records attributed to A Bank.

At least 91 records · Page 5Linked to original sources
PubMed1987

Human beta-globin gene expression after gene transfer using retroviral vectors.

A retroviral vector containing a 4.4-kb Pst I human beta S-globin gene and a neomycin resistance gene was used to infect NIH-3T3 and mouse erythroleukemia cells (MELC). In MELC, human beta-globin mRNA transcripts are transcribed and properly initiated and spliced. In some cases, there is an appropriate increase in beta-globin mRNA on addition of dimethylsulfoxide (DMSO), an inducer of hemoglobin synthesis and erythroid differentiation in these cells. When NIH-3T3 cells are infected with the same retroviral vector, there is less globin mRNA accumulation and no evidence for appropriate regulation. Human beta-globin gene expression in MELC clones induced with DMSO is 2-3% that of endogenous mouse beta-globin gene expression. These results indicate that retroviral vectors can be used to transfer and appropriately express human beta-globin genes in erythroid cells.

Cells, Cultured↗
PubMed1987

Regulated expression of amplified human beta globin genes.

Gene therapy for the beta thalassemias and sickle cell anemia will require high levels of expression of human beta globin genes. One method to achieve this goal is amplification of globin genes transferred into the stem cells in the bone marrow of these patients. If the amplified genes remain normally regulated, they will then further increase their expression on being induced to differentiate along an erythroid pathway. To begin this study, we constructed a plasmid containing a neomycin resistance gene, a human beta globin gene, and a wild-type DHFR cDNA, and transfected it into mouse erythroleukemia cells. All the G418-resistant clones analyzed acquired and expressed the human beta globin gene. By serial passage of the cells in increasing concentrations of methotrexate, the exogenous human beta globin genes were stably amplified in all lines, and all increased their globin mRNA expression roughly proportional to their augmented copy number. Most of the clones further increased their beta globin expression on addition of an erythroid stimulus (dimethylsulfoxide). These results indicate that globin gene amplification may be useful in increasing globin mRNA expression in further experiments whose goal is gene therapy.

Clone Cells↗
PubMed1987

CML patients in blast crisis have breakpoints localized to a specific region of the BCR.

Chronic myelogenous leukemia (CML) is associated with the Philadelphia (Ph) chromosome, which results from a reciprocal translocation between chromosomes 9 and 22. This activates the abl oncogene by moving it from chromosome 9 and combining it with sequence located on chromosome 22. The new fusion gene, with chromosome 22 sequence at its 5' end and chromosome 9-abl sequence at its 3' end, generates a new messenger RNA (mRNA) and protein that are implicated in the pathogenesis of CML. The breakpoint near the c-abl locus on chromosome 9 can occur within a large area. In contrast, the breakpoints on chromosome 22 are concentrated within a 6 kilobase (kb) region termed the breakpoint cluster region (bcr). This study was designed to determine whether chronic-phase and blast crisis patients had identifiable differences in the structure of their Ph chromosomes. Restriction mapping of the chromosome 22 translocation breakpoints performed for 26 patients showed that the breakpoints of eight of the nine patients in blast crisis were in the 3' portion of the bcr, whereas the breakpoints in the 17 patients in the chronic phase were clustered in the 5' portion of the bcr. This suggests a strong correlation between a 3' bcr breakpoint and blast crisis in CML.

Blast Crisis↗
PubMed1987

Human globin gene expression after gene transfer.

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.

Anemia, Sickle Cell↗
PubMed1987

Beta-thalassemia syndromes.

In summary, the beta-thalassemias are due to defects in or around the structural beta-globin gene. In some Indian patients, there is deletion of sequence at the 3' end of the beta-globin gene. Most commonly, single nucleotide mutations cause beta(+)- and beta(0) -thalassemia. More than 30 such mutations have been identified. Defects in the promoter region 5' to the gene as far 5' as -87 and closer to the gene at -27 and -28 in the ATA sequence can cause beta (+)-thalassemia. Single nucleotide changes in coding regions leading to termination or nonsense codons commonly cause beta (0)-thalassemia. In addition, beta(0)-thalassemia can be due to single nucleotide changes in the invariant GT at the 5' splice junction in IVS 1 and 2 and in the AG at the 3' end of IVS 2. Additionally, single nucleotide mutations can occur within IVS that result in both beta(+)- and beta(0)-thalassemia. New splice sites are usually the result of these single nucleotide mutations, and they lead to new, abnormal splicing patterns. In some instances, beta (+)-thalassemia results when a new splice signal created within IVS is still associated with some continued normal splicing as well as with abnormal splicing. The abnormal splicing leads to abnormal mRNA, while the normal splicing leads to some normal mRNA and the beta (+)-pheno-single In other cases, such as with the defect as position 705 of IVS 2, the single nucleotide change within the IVS allows only abnormal mRNA splicing, and it results in beta (0)-thalassemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1986

Beta thalassemia due to a novel mutation in IVS 1 sequence donor site consensus sequence creating a restriction site.

During a systematic screening of Algerian thalassemics by determining the DNA polymorphism haplotypes in the beta globin gene cluster, a novel haplotype was identified. The DNA of a homozygous individual was cloned and sequenced. The mutation, a G----A change, at position 5 of the small intervening sequence, probably interferes with normal splicing events, and, moreover, creates a new Eco RV restriction site that provides a useful diagnostic tool for detecting this condition.

Base Sequence↗
PubMed1986

Frequent and extensive deletion during the 9,22 translocation in CML.

Chromosomal translocation is one mechanism by which cellular oncogenes may be activated during tumorigenesis. The translocation of the abl oncogene to the Philadelphia chromosome in chronic myelogenous leukemia (CML) results in a new RNA transcript that fuses sequence from chromosome 22 to sequence from the abl oncogene. This RNA presumably codes for a new abl-related protein product found in CML, the activity of which is different from the normal abl protein. The molecular structure of the translocation varies from patient to patient, and the individual variation in RNA transcript and protein product remains to be defined. This report describes the frequent occurrence of chromosomal deletion within the 9q+ chromosome during these translocations. The location of the deletions suggests that some mechanism maintains the chromosomal breakpoint on the Philadelphia chromosome within a limited region. These deletions complicate the interpretation of Southern blots as a means of detecting the translocation.

Chromosome Deletion↗
PubMed1986

Expression of a cloned Lepore globin gene.

Lepore globin is synthesized in markedly diminished amounts (approximately 10% to 15% of normal beta-globin) in human erythroid cells. To study the molecular mechanisms responsible for the diminished biosynthesis of Lepore globin, the Lepore-Boston gene was cloned from a charon phage DNA library and expressed in HeLa cells. Northern blotting and S1 nuclease analyses indicated that the Lepore gene produced less globin mRNA than a beta-gene and more than a delta-gene. The results indicate that expression of the Lepore-Boston gene in HeLa cells is reduced to an extent comparable to that seen in erythroid precursors in vivo. This indicates that the decrease in Lepore globin gene transcription is due to the delta-nucleotide sequences either in the 5' flanking region or within this gene.

Base Sequence↗
PubMed1985

Reversibility of IVS 2 missplicing in a mutant human beta-globin gene.

We have studied the aberrant splicing of a human beta thalassemia globin gene by expression of the cloned gene in HeLa cells and oligomer-directed mutagenesis. A mutation 705 nucleotides into the large intervening sequence (IVS 2) of this gene leads to missplicing in which IVS 2 is incompletely removed, via two aberrant splices, from the vast majority of transcripts. One splice is from the 5' end of IVS 2 to a normal sequence 580 nucleotides into IVS 2 and another is from the mutated site 705 nucleotides into IVS 2 to the 3' end of the IVS. To study the splicing of this gene further, a mutation was introduced into the cryptic 3' splice site at position 580. This results in the complete removal of IVS 2 despite the presence of the thalassemia mutation at 705. The reversal of abnormal splicing by a change in the cryptic splice site suggests that the two abnormal splices are subtly interdependent. Thus, single base changes within IVS 2 can drastically alter the pattern of splicing in a human beta-globin gene.

Base Sequence↗
PubMed1985

DNA sequences regulating human beta globin gene expression.

Human delta globin is expressed at approximately 1-2% of the level of human beta globin in erythroid cells despite the marked homology between these two globins. To determine the DNA sequences responsible for this effect, delta and beta globin genes and fusion products of these genes constructed in vitro were transfected and expressed in HeLa cells. The results indicate that when the small intervening sequence of the beta gene (beta IVS 1) is replaced by delta IVS 1, expression of the chimeric gene is the same as that of the normal beta globin gene. By contrast, when the large intervening sequence of the beta gene (beta IVS 2) is replaced by delta IVS 2, expression of the chimeric gene is markedly reduced. These results suggest that there are signals within IVS 2 of the delta and beta genes which affect their relative expression.

Base Sequence↗
PubMed1985

Tissue specific transcription of the human epsilon-globin gene following transfection into the embryonic erythroid cell line K562.

We have introduced a plasmid containing the human epsilon-globin gene either stably or transiently into a number of erythroid or non-erythroid cell lines, and analysed the accuracy and efficiency of transcription. In non-erythroid cells (or in mouse erythroleukaemia (MEL) cells in which adult but not embryonic globin genes are expressed) transcription of the epsilon-globin gene occurs mainly from a site 200 bp upstream of the major cap site (the -200 cap site). In the human K562 cell line, in which the endogenous epsilon-globin gene is transcribed at high levels, transcription initiation from the introduced gene occurs mainly from the major cap site. Transcriptional activity of the epsilon-globin gene introduced into K562 cell is quantitatively similar to that of the endogenous gene. This suggests the presence (or absence) in K562 cells of factor(s) which activate (or repress) the epsilon-globin gene in a tissue specific manner.

Cell Line↗
PubMed1985

Trans acting regulation of beta globin gene expression in erythroleukemia (K562) cells.

K562 cells are induced by hemin to produce gamma and epsilon globin but not beta globin, although the beta globin gene is intact, and when isolated is expressed in a transient expression assay (1, 2). We have previously shown that an epsilon globin gene transferred into K562 cells is expressed and inducible (3). In this paper, we report the stable transfer of a sickle or betaS globin gene into K562 cells. Thirty-six different transformed lines were tested; 24 of 36 lines contained an intact betaS globin gene. However, using S1 nuclease, Dot blot, and Northern blotting analyses, none of these lines showed beta globin mRNA expression. These results indicate that trans acting factors are responsible for the lack of expression of the beta globin gene in K562 cells.

Cell Line↗
PubMed1985

Abnormal globin gene structure and expression in beta-thalassemia.

Over the past five years, several new defects in the beta-thalassemias have been described from this laboratory using both restriction enzyme and sequencing analyses of cloned beta-thalassemia genes. The enzyme HphI has been shown to recognize a single nucleotide change at the 5' end of beta-IVS 2, and, using restriction enzyme analysis, demonstrated for the first time a specific defect associated with beta(0)-thalassemia. Cloning and sequencing of a beta-thalassemia gene have identified a single base change within IVS 2 at a position 705 nucleotides from the 5' end of IVS 2 that results in a beta(0)-thalassemia phenotype; no normal splicing occurs in this gene despite the fact that both the 5' and 3' ends of IVS 2 are unchanged. A unique and strong cryptic 3' acceptor splice site present in the normal gene at a position 580 nucleotides from the 5' end is used extensively in the mutant gene. Studies of this gene have indicated that there are sequences within IVS that are responsible for optimal expression of this gene; changes in these sequences can lead to markedly abnormal patterns of splicing. In addition, beta-globin gene expression has been evaluated in human erythroleukemia cells, K562 cells, and, although stable transformants with integrated beta-globin genes have been obtained, none of these transformants expressed the added beta-globin genes. This is presumably due to trans-acting factors or distal cis-acting effects that suppress the expression of these added beta-globin genes. In addition, a low epsilon-producing cell line, Bos cells, was used as a recipient for an exogenous epsilon-globin gene. A neomycin resistance gene was cotransfected into these cells, and a neomycin analogue (G418) was used to select cells containing both the neomycin resistance and epsilon-globin genes. Using Southern blotting, 10 of 11 stably transformed G418-resistant lines, which contain intact epsilon-globin genes, express epsilon-globin mRNA at much higher levels than the Bos cells into which they were transfected. Two of these lines express the epsilon-globin genes at a level comparable to that of wild-type K562 cells. These results indicate that the transfer and expression of human globin genes in human erythroid cells is feasible, and can occur at a high level.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Line↗
PubMed1985

Increased expression of a novel c-abl-related RNA in K562 cells.

The c-abl locus is translocated from chromosome 9 to chromosome 22 in chronic myelogenous leukemia (CML), creating the Philadelphia chromosome (22q-, Ph1), one of the most consistent chromosomal abnormalities found in human hematologic malignancy. The K562 cell line is a human cell line originally derived from a patient with CML. We have isolated cloned human c-abl probes to analyze the organization and expression of abl genes in patients with CML and in K562 cells. With these probes, we confirm the amplification of abl genes in K562 cells. In addition, we demonstrate the presence of increased amounts of a novel RNA species hybridizing to a c-abl probe in K562 cells. This same large RNA species is present in addition to two normal transcripts in the leukemic cells of patients with CML. These results provide evidence that the c-abl locus is abnormally expressed in CML.

Cell Line↗
PubMed1985

Variable breakpoints on the Philadelphia chromosome in chronic myelogenous leukemia.

The abl oncogene is translocated from chromosome 9 to 22 in the creation of the Philadelphia (Ph1) chromosome. This article describes new translocation breakpoints identified in two patients with chronic myelogenous leukemia using Southern blotting and cloned human DNA probes from chromosome 9. The translocation breakpoints on chromosome 9 in both of these patients lie closer to the human cellular abl (c-abl) gene, and the chromosome 22 breakpoints are distributed more widely than previously reported. These data help to define more clearly the chromosomal span of the breakpoints and indicate that some translocations include very little chromosome 9 sequence located 5' to the c-abl gene.

Chromosome Aberrations↗