scholarly journals Identification of a recently evolved goat embryonic beta-globin pseudogene which retains transcriptional activity in vitro.

1984 ◽  
Vol 4 (10) ◽  
pp. 2120-2127 ◽  
Author(s):  
S G Shapiro ◽  
J B Lingrel
Keyword(s):  
Transcriptional Activity ◽  
Stop Codon ◽  
Globin Gene ◽  
Consensus Sequence ◽  
Duplication Event ◽  
Beta Globin ◽  
Gene Sets ◽  
Sequence Elements ◽  
Group 5

A clone containing the entire goat epsilon V beta-globin gene, which lies downstream from the two tandemly duplicated four-gene sets containing the beta C and beta A genes in the linkage group 5'-epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-3', was isolated, and the sequence of the gene was determined. epsilon V is most homologous to the first gene in each of these sets, epsilon I and epsilon III, and appears to be a third duplicated copy of these genes, possibly the first gene in a third four-gene set. Homology of epsilon V to epsilon I is very high (93.2%) in coding regions, and all transcription, processing, and potential translation consensus sequence elements appear to be present, although the Hogness box of epsilon V is altered compared with that of epsilon I by the deletion of an A(AATAAAA----AATAAA). Nevertheless, epsilon V is clearly a pseudogene as a result of two deletions and one insertion (or insertion-deletion) in its coding sequence, the first of which produces an in-frame stop codon at amino acid 54. Unlike the more highly mutated goat beta-like pseudogene duplicates psi beta X and psi beta Z, epsilon V acquired its defects after the duplication event in which it was created. Its recently acquired defects have left the epsilon V promoter sufficiently conserved to retain transcriptional activity in vitro. The acquisition of defects by this gene may be related to the multiple gene duplications which have created at least five epsilon type genes in the goat beta-globin locus.

Identification of a recently evolved goat embryonic beta-globin pseudogene which retains transcriptional activity in vitro

1984 ◽  
Vol 4 (10) ◽  
pp. 2120-2127
Author(s):  
S G Shapiro ◽  
J B Lingrel
Keyword(s):  
Transcriptional Activity ◽  
Stop Codon ◽  
Globin Gene ◽  
Consensus Sequence ◽  
Duplication Event ◽  
Beta Globin ◽  
Gene Sets ◽  
Sequence Elements ◽  
Group 5

A clone containing the entire goat epsilon V beta-globin gene, which lies downstream from the two tandemly duplicated four-gene sets containing the beta C and beta A genes in the linkage group 5'-epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-3', was isolated, and the sequence of the gene was determined. epsilon V is most homologous to the first gene in each of these sets, epsilon I and epsilon III, and appears to be a third duplicated copy of these genes, possibly the first gene in a third four-gene set. Homology of epsilon V to epsilon I is very high (93.2%) in coding regions, and all transcription, processing, and potential translation consensus sequence elements appear to be present, although the Hogness box of epsilon V is altered compared with that of epsilon I by the deletion of an A(AATAAAA----AATAAA). Nevertheless, epsilon V is clearly a pseudogene as a result of two deletions and one insertion (or insertion-deletion) in its coding sequence, the first of which produces an in-frame stop codon at amino acid 54. Unlike the more highly mutated goat beta-like pseudogene duplicates psi beta X and psi beta Z, epsilon V acquired its defects after the duplication event in which it was created. Its recently acquired defects have left the epsilon V promoter sufficiently conserved to retain transcriptional activity in vitro. The acquisition of defects by this gene may be related to the multiple gene duplications which have created at least five epsilon type genes in the goat beta-globin locus.

scholarly journals Serum factors can modulate the developmental clock of gamma- to beta-globin gene switching in somatic cell hybrids.

1993 ◽  
Vol 13 (8) ◽  
pp. 4844-4851 ◽  
Author(s):  
G Zitnik ◽  
Q Li ◽  
G Stamatoyannopoulos ◽  
T Papayannopoulou
Keyword(s):  
Globin Gene ◽  
Cumulative Number ◽  
Beta Globin ◽  
Serum Factors ◽  
Beta Globin Gene ◽  
Cell Divisions ◽  
Growth Promoting ◽  
Gene Switching ◽  
Mouse Erythroleukemia

The fusion of human fetal erythroid (HFE) cells with mouse erythroleukemia (MEL) cells produces stable synkaryons (HFE x MEL) which can be monitored for extended periods of time in culture. Initially these hybrids express a human fetal globin program (gamma >> beta), but after weeks or months in culture, they switch to an adult pattern of globin expression (beta >> gamma). The rate at which hybrids switch to the adult phenotype is roughly dependent on the gestational age of the fetal erythroid cells used in the fusion, suggesting that the rate of switching in vitro may be determined by a developmental clock type of mechanism, possibly involving the cumulative number of divisions experienced by the human fetal cells. To investigate whether the number or rate of cell divisions postfusion can influence the rate of switching, we monitored the rate of switching in hybrids from independent fusions under growth-promoting (serum-replete) and growth-suppressing (serum-deprived) conditions. We found that hybrids grown under serum-deprived or serumless conditions switched more rapidly to adult globin expression than did their counterparts in serum-replete conditions. Neither the number of cumulative cell divisions nor time in culture per se predicted the rate of switching in vitro. Our data suggest that factors present in serum either retard switching of hybrids by their presence or promote switching by their absence, indicating that globin switching in vitro can be modulated by the environment; however, once switching in HFE x MEL hybrids is complete, serum factors cannot reverse this process.

Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression

1985 ◽  
Vol 5 (4) ◽  
pp. 649-658
Author(s):  
G M Veldman ◽  
S Lupton ◽  
R Kamen
Keyword(s):  
Dna Replication ◽  
Globin Gene ◽  
Mrna Levels ◽  
Beta Globin ◽  
Transcriptional Enhancer ◽  
Globin Mrna ◽  
Enhancer Region ◽  
Sequence Elements ◽  
Multiple Copies ◽  
A Site

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.

scholarly journals The inactive beta globin gene on a gamma delta beta thalassemia chromosome has a normal structure and functions normally in vitro

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 766-770
Author(s):  
PT Curtin ◽  
YW Kan
Keyword(s):  
Globin Gene ◽  
Large Deletion ◽  
Beta Thalassemia ◽  
Ribonuclease Protection Assay ◽  
Beta Globin ◽  
Gamma Delta ◽  
Beta Globin Gene ◽  
Thalassemia Minor

We have previously described an English family with gamma delta beta- thalassemia in which a large deletion stops 25 kilobases (kb) upstream from the beta-globin gene locus, and yet the beta-globin gene is inactive in vivo. Affected family members had a beta-thalassemia minor phenotype with a normal hemoglobin A2 level. Gene mapping showed that these subjects were heterozygous for a chromosome bearing a large deletion that began in the G gamma-globin gene, extended through the epsilon-globin gene, and continued upstream for at least 75 kb. The A gamma-, delta-, and beta-globin gene loci on this chromosome were intact. To examine the possibility that an additional defect was present in the beta-globin gene, we cloned, sequenced, and examined the expression of the beta-globin gene from the affected chromosome. No mutation was found in the beta-globin gene sequence from 990 base-pairs 5′ to the cap site to 350 basepairs 3′ to the polyadenylation signal. The gene was subcloned into an expression vector and introduced into HeLa cells. Analysis of RNA derived from these cells, using a ribonuclease protection assay, revealed qualitatively and quantitatively normal transcription. Thus a structurally and functionally normal beta-globin gene is inactive in the presence of a large deletion more than 25 kb upstream. The loss of beta-globin gene function may be due to disturbance of chromatin conformation caused by the deletion or may be the result of loss of upstream sequences that are necessary for beta-globin gene expression in vivo.

scholarly journals Characterization of the DNase I hypersensitive site 3' of the human beta globin gene domain

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2781-2790
Author(s):  
DE Fleenor ◽  
RE Kaufman
Keyword(s):  
Topoisomerase Ii ◽  
Globin Gene ◽  
Dnase I ◽  
Globin Genes ◽  
Beta Globin ◽  
Hypersensitive Site ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Beta Globin Gene

The members of the human beta globin gene family are flanked by strong DNase I hypersensitive sites. The collection of sites 5' to the epsilon globin gene is able to confer high levels of expression of linked globin genes, but a function has not been assigned to the site 3' to the beta globin gene (3'HS1). Our analysis of this DNase I super hypersensitive site shows that the region is composed of multiple DNase I sites. By examination of the DNA sequence, we have determined that the region is very A/T-rich and contains topoisomerase II recognition sequences, as well as several consensus binding motifs for GATA-1 and AP-1/NF-E2. Gel mobility shift assays indicate that the region can interact in vitro with GATA-1 and AP-1/NF-E2, and functional studies show that the region serves as a scaffold attachment region in both erythroid and nonerythroid cell lines. Whereas many of the physical features of 3'HS1 are shared by 5'HS2 (a component of the 5' locus control region), transient expression studies show that 3' HS1 does not share the erythroid-specific enhancer activity exhibited by 5'HS2.

scholarly journals A novel deletion of approximately 27 kb including the beta-globin gene and the locus control region 3'HS-1 regulatory sequence: beta zero- thalassemia or hereditary persistence of fetal hemoglobin?

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 822-827 ◽  
Author(s):  
AJ Dimovski ◽  
V Divoky ◽  
AD Adekile ◽  
E Baysal ◽  
JB Wilson ◽  
...  
Keyword(s):  
Control Region ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Locus Control Region ◽  
Regulatory Sequence ◽  
Beta Globin ◽  
Gamma Chain ◽  
Beta Globin Gene ◽  
Gamma Globin

Abstract A novel deletion of approximately 27 kb with the 5′ breakpoint 1.5 to 2.2 kb upstream of the beta-globin gene, and the 3′ breakpoint approximately 24 kb downstream of the beta-globin gene, has been found in five members of two families from Southeast Asia (Vietnam and Cambodia). Six members of another family from China, previously reported from our laboratory, have also been shown to carry this deletion. The patients presented with mild hypochromia and microcytosis, a hemoglobin (Hb) A2 level of approximately 4.0%, and a markedly increased, heterocellularly distributed, Hb F level (14.0 to 26.0%). In vitro globin-chain synthesis showed a mild imbalance with appreciable gamma-chain compensation (alpha/beta + gamma ratio of 1.46). The 3′ end of this deletion includes the 3′HS-1, and we hypothesize that removal of this region results in the loss of its gamma-globin gene-silencing effect, which causes a markedly elevated Hb F level with a modest increase in Hb A2 levels, unlike the situation in other deletional beta zero-thalassemias. The possible influence of particular sequence variations in the locus control region 5′HS-2 and the G gamma promoter, present on the chromosome with this deletion, on the overall gamma-globin gene should also be considered.

scholarly journals A simple approach to prenatal diagnosis of beta-thalassemia in a geographic area where multiple mutations occur

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1357-1360 ◽  
Author(s):  
SP Cai ◽  
JZ Zhang ◽  
DH Huang ◽  
ZX Wang ◽  
YW Kan
Keyword(s):  
Southern China ◽  
Globin Gene ◽  
Blot Hybridization ◽  
Geographic Area ◽  
Simple Approach ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Dot Blot ◽  
Polymerase Chain

Abstract We describe a simple approach for detecting beta-thalassemia mutations in geographic areas such as southern China where multiple mutations are known to occur. Segments of the beta-globin gene were amplified in vitro by using the polymerase chain reaction. Dot blot hybridization of the amplified DNA with oligonucleotide probes corresponding to the six mutations found in southern China could directly identify the mutations causing beta-thalassemia in the affected families. The increased number of target sequences after amplification allows the use of 35S-labeled probes, which are reusable for up to 3 months. The mutations can be determined in two days.

scholarly journals Clinical and genetic heterogeneity in black patients with homozygous beta-thalassemia from the southeastern United States

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 1007-1014 ◽  
Author(s):  
JM Gonzalez-Redondo ◽  
TA Stoming ◽  
KD Lanclos ◽  
YC Gu ◽  
A Kutlar ◽  
...  
Keyword(s):  
South Carolina ◽  
Genomic Dna ◽  
Stop Codon ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Nucleotide Substitutions ◽  
Taq Polymerase ◽  
Black Patients ◽  
Codon 61

Abstract The presence of various substitutions and deletions resulting in beta- thalassemia was studied in 19 black patients with homozygous beta- thalassemia and in numerous relatives; all patients were from Georgia, South Carolina, and Alabama. Methodology included gene mapping, amplification of genomic DNA with Taq polymerase, identification of known nucleotide substitutions or a single nucleotide deletion through hybridization with synthetic oligonucleotides, cloning and sequencing of a beta-globin gene, and sequencing of amplified genomic DNA. Of the 38 chromosomes tested, 21 (55%) had the A----G substitution at nt -29, eight (21%) had the C----T substitution at nt -88, three (8%) had the substitution at codon 24, while one each of the following abnormalities were also detected: frameshift at codon 6, a C----A mutation at nt 848 of the beta IVS-II (new), an A----T mutation at codon 61 (new), a deletion of 1.35 kilobases including the 5′ end of beta, a Ggamma(Agammadelta beta) degree-thalassemia, and one thalassemia determinant that remained unidentified. The C----A mutation at nt 848 of IVS-II occurred at a position 3 nucleotides 5′ to the third exon, adjacent to the invariant AG dinucleotide of the acceptor sequence. The A----T mutation in codon 61 (AAG----TAG) resulted in the creation of a stop codon and thus in beta degree-thalassemia. The various mutations occurred on chromosomes with different haplotypes; however, chromosomes with a specific mutation but with different haplotypes belonged to one specific framework, which suggested that crossovers were responsible for these different types. Hemoglobin (Hb) F levels were generally high (55% to 75% with 98.5% in one patient with beta degree/beta degree); a few patients with specific haplotypes and an alpha-thalassemia-2 heterozygosity had a lower Hb F level. The Ggamma in the Hb F was consistently high when the C----T mutation occurred at nt -158 to the Cap site of the Ggamma-globin gene; seven patients with +/+ at this site had an average Ggamma of 73.8%, eight patients with +/- had 64.8%, and one patient with -/- had 34.2%. Variations in hematologic values and in Hb F, Ggamma, and Hb A2 levels of relatives with a beta- thalassemia heterozygosity depended to some extent on the types of mutations or deletions and on the haplotypes of the chromosomes with the beta-thalassemia determinant.

scholarly journals Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro.

1988 ◽  
Vol 8 (10) ◽  
pp. 4484-4491 ◽  
Author(s):  
A Mayeda ◽  
Y Ohshima
Keyword(s):  
Consensus Sequence ◽  
Donor Site ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Beta Globin ◽  
Vitro Assay ◽  
Active Donor

We constructed SP6-human beta-globin derivative plasmids that included possible donor site (5' splice site) sequences at a specified position within the first intron. The runoff transcripts from these templates truncated in the second exon were examined for splicing in a nuclear extract from HeLa cells. In addition to the products from the authentic donor site, a corresponding set of novel products from the inserted, alternative donor site was generated. Thus, a short sequence inserted within an intron can be an active donor site signal in the presence of an authentic donor site. The active donor site sequences included a 9-nucleotide consensus sequence, 14- or 16-nucleotide sequences at the human beta-globin first or second donor, and those at simian virus 40 large T antigen or small t antigen donor. These included 3 to 8 nucleotides of an exon and 6 to 8 nucleotides of an intron. The activity of the inserted donor site relative to that of the authentic donor site depended on the donor sequence inserted. The relative activity also strongly depended on the concentrations of both KCl (40 to 100 mM) and MgCl2 (1.6 to 6.4 mM). At the higher KCl concentrations tested, all the inserted, or proximate, donor sites were more efficiently used. Under several conditions, some inserted donor sites were more active than was the authentic donor site. Our system provides an in vitro assay for donor site activity of a sequence to be tested.

scholarly journals Transcriptional promiscuity of the human alpha-globin gene.

1989 ◽  
Vol 9 (1) ◽  
pp. 241-251 ◽  
Author(s):  
E Whitelaw ◽  
P Hogben ◽  
O Hanscombe ◽  
N J Proudfoot
Keyword(s):  
Cell Lines ◽  
Transcriptional Activity ◽  
Globin Gene ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Erythroid Cell ◽  
Alpha Globin ◽  
Multiple Copies ◽  
Positive Effect

The human alpha-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SP1-binding site. We have investigated whether there is a relationship between these two observations. First, we investigated the mouse alpha-globin gene since it does not lie in an HTF island. We have demonstrated that it was not transcriptionally promiscuous. Second, we studied the transcriptional activity of the human alpha-globin gene in the absence of the GC-rich region containing putative SP1-binding sites and found a small (two- to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human alpha-globin gene. We found that in a nonreplicating system, the human alpha-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since we only observed enhancer independence of the human alpha-globin gene in a high-copy-number replicating system, we suggest that competition for trans-acting factors could explain these results. Finally, our experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human alpha-globin cap site or within the gene itself.

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