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C Dobkin

Publications and source records attributed to C Dobkin.

At least 19 recordsLinked to original sources
PubMed1987

Expression of human gamma-globin genes in human erythroleukemia (K562) cells.

K562 cells express embryonic (epsilon) and fetal (gamma) globins and hemoglobins but not adult (beta) globin. To define the cis acting regulatory elements involved in the discrimination between gamma and beta genes, we have constructed chimeric genes composed of portions of gamma and beta and evaluated their expression in stable K562 transfectants. A gamma beta fusion gene containing gamma 5' sequences to the EcoRI site in exon 3 and beta sequences 3' is expressed at 10-40% that of the endogenous gamma level. In 50% of the lines, this fusion gene appropriately increases its expression in response to hemin, an inducer of endogenous globin gene expression in K562 cells. In contrast, a beta gamma fusion gene, containing beta sequences 5' to the EcoRI site in exon 3 and gamma sequences 3', is neither expressed nor correctly initiated. A beta gene containing gamma-intervening sequence (IVS) 2 accumulates an mRNA transcript when analyzed with a 3' beta probe. However, no correctly initiated beta mRNA is observed. A gamma gene with beta-IVS 2 is only inducible in one of six expressing clones. All the results are consistent with the presence of stage-specific trans acting factors in K562 cells that stimulate expression of gamma genes and suggest a significant role for gamma-IVS 2 in gamma gene expression.

Cell Line↗
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

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

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

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

Structure and expression of two beta genes in a beta thalassemia homozygote.

Two beta globin gene alleles have been cloned and characterized from a patient with beta + thalassemia. Both beta genes have single base mutations in the small intervening sequence (IVS 1); one 6 nucleotides and the other 110 nucleotides from the 5' end of IVS 1. Both genes lead to abnormal splicing of beta globin mRNA precursors when expressed in HeLa cells. Despite the fact that both alleles produce some normal beta globin mRNA transcripts, the patient has clinically severe beta + thalassemia (Cooley's anemia).

Base Sequence↗
PubMed1984

Erythroleukemia (K562) cells contain a functional beta-globin gene.

K562 cells are human erythroid cells that synthesize embryonic and fetal globins but not adult beta-globin. A cloned beta-globin gene was isolated from K562 cells and transfected into HeLa cells. The RNA transcripts produced were comparable in both amount and size to those obtained with a normal beta-globin gene.

Cloning, Molecular↗
PubMed1983

Terminal adenylation in the synthesis of RNA by Q beta replicase.

We investigated the apparent requirement that Q beta replicase must add a nontemplated adenosine to the 3' end of newly synthesized RNA strands. We used abbreviated MDV-1 (+)-RNA templates that lacked either 62 or 63 nucleotides at their 5' end in Q beta replicase reactions. The MDV-1 (-)-RNA strands synthesized from these abbreviated (+)-strand templates were released from the replication complex, yet they did not possess a nontemplated 3'-terminal adenosine. These results imply that, despite observations that all naturally occurring RNAs synthesized by Q beta replicase possess a nontemplated 3'-adenosine, the addition of an extra adenosine is not an obligate step for the release of completed strands. Since the abbreviated templates lacked a normal 5' end, it is probable that a particular sequence at the 5' end of the template is required for terminal adenylation to occur.

Base Sequence↗
PubMed1983

Three regions upstream from the cap site are required for efficient and accurate transcription of the rabbit beta-globin gene in mouse 3T6 cells.

Cloned rabbit beta-globin genes, modified in vitro by restructuring or site-directed mutagenesis, were introduced into mouse 3T6 cells, and the resulting transcripts were analyzed by nuclease S1 mapping. The first 109 bp preceding the cap site sufficed for maximal beta-globin transcription. This segment contained three functionally important regions of the ATA box region; the CCAAT box region; and the -100 region. The latter consists of an imperfect tandemly repeated sequence of 14 bp and 15 bp, both copies of which are required for optimal promoter function. Each of three regions contains two or more nucleotide positions where the introduction of point mutations reduces transcription by at least a factor of 2.

Animals↗