scholarly journals Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes.

1985 ◽  
Vol 82 (24) ◽  
pp. 8349-8353 ◽  
Author(s):  
R. A. Padgett ◽  
M. M. Konarska ◽  
M. Aebi ◽  
H. Hornig ◽  
C. Weissmann ◽  
...  
Keyword(s):  
Globin Genes ◽  
Beta Globin ◽  
Branch Site ◽  
In Vitro Splicing

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.

In vitro splicing pathways of pre-mRNAs containing multiple intervening sequences?

1987 ◽  
Vol 7 (10) ◽  
pp. 3428-3437
Author(s):  
K M Lang ◽  
R A Spritz
Keyword(s):  
Temporal Order ◽  
Exon Skipping ◽  
Beta Globin ◽  
Splice Site Selection ◽  
Interleukin 3 ◽  
Intervening Sequences ◽  
In Vitro Splicing ◽  
Lag Times ◽  
Alpha Lactalbumin

We analyzed the in vitro splicing pathways of three multi-intervening-sequence (IVS) pre-mRNAs: human beta-globin, which contains two IVSs (K. M. Lang, V. L. van Santen, and R. A. Spritz, EMBO J. 4:1991-1996, 1985); rat alpha-lactalbumin, which contains three IVSs; and murine interleukin-3, which contains four IVSs. We found that there are highly preferred pathways of IVS removal from these multi-IVS pre-mRNAs in vitro. The three IVSs of rat alpha-lactalbumin pre-mRNA were excised sequentially from 5' to 3'; in most molecules, IVS1 was removed first, followed by IVS2 and finally by IVS3. The splicing pathway of interleukin-3 pre-mRNA in vitro was more complex. The four IVSs were excised in a highly preferred temporal order, but the order was not strictly sequential or directional. In most molecules, IVS1 and IVS4 were removed first, either simultaneously or in rapid succession. Subsequently, IVS2 was excised, followed by IVS3. The observed splicing pathways apparently resulted from differences in lag times and maximum excision rates of the different IVSs. We detected no exon skipping during splicing of these transcripts in vitro. These observations have implication for proposed models of splice site selection.

scholarly journals Constitutive expression of GATA-1, EPOR, alpha-globin, and gamma-globin genes in myeloid clonogenic cells from juvenile chronic myelocytic leukemia

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 323-328 ◽  
Author(s):  
E Privitera ◽  
R Schiro ◽  
D Longoni ◽  
A Ronchi ◽  
A Rambaldi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemic Cells ◽  
Globin Genes ◽  
Chronic Myelocytic Leukemia ◽  
Beta Globin ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Myelocytic Leukemia ◽  
Alpha Globin

Juvenile chronic myelocytic leukemia (JCML) is a rare disorder of early childhood. Characteristic of JCML are the progressive appearance of high levels of fetal hemoglobin (HbF), reflecting a true reversion to a fetal type of erythropoiesis, and the presence of colony-forming cells able to grow in vitro spontaneously in the absence of growth factors. To better understand the relationship between the erythroid abnormalities and the leukemic process, we analyzed the expression pattern of specific genes related to erythroid differentiation--GATA-1, EPOR, alpha-globin, beta-globin, and gamma-globin genes--in JCML peripheral blood (PB) cells and in vitro-derived colonies. Northern blot analysis of PB cells from five JCML patients indicated levels of GATA-1 transcripts much higher than those usually found in other types of leukemic cells, and S1 nuclease protection assay detected significantly increased expression of gamma-globin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of single granulocyte-macrophage colony-forming unit (CFU-GM) colonies, obtained in vitro in the absence of added growth factors from four JCML patients, detected GATA-1, EPOR, and globin (alpha and gamma) transcripts in most of the colonies tested, in contrast with control CFU-GM from normal bone marrow, which were positive only for GATA-1. Single JCML colonies were tested for the presence of two different transcripts; whereas alpha- and gamma-globin genes appeared mostly coexpressed, beta-globin mRNA was detected only in a minority of the gamma-globin-positive colonies, indicating that the leukemic pattern of hemoglobin synthesis is mainly fetal. In addition, the leukemic cells occurring during blast crisis of one of our patients displayed the typical features of a stem cell leukemia (CD34+, CD19-, CD2-, myeloperoxidase-). In this sorted CD34+ population, we detected the presence of a marker chromosome, der(12)t(3;12), previously identified in bone marrow cells at diagnosis and an expression pattern superimposable to that of the JCML colonies, consistently displaying a high gamma-globin:beta-globin mRNA ratio. The expression of erythroid markers within populations of leukemic cells, both in vivo and in vitro, supports the hypothesis that abnormal JCML erythroid cells may originate from the same mutated progenitor that sustains the growth of the leukemic cells.

A Deletion Of About 11kb Starting From 0.5 Kb 5’ To The β-Globin Gene Reactivates Foetal Haemoglobin and Leads To β Thalassemia Intermedia When Associated To a β-Zero Thalassemic Mutation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4702-4702
Author(s):  
Photis Beris ◽  
Tanguy Araud ◽  
Lorella Clerici ◽  
Anne-Pascale Grandjean ◽  
Georgios Georgiou ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Globin Gene ◽  
Thalassemia Major ◽  
Globin Genes ◽  
Beta Globin ◽  
Thalassemia Intermedia ◽  
Chromosome 11 ◽  
Exact Size ◽  
Hg19 Assembly

Background and Aims Thalassemia intermedia is characterized by severe but not transfusion dependent anemia secondary to seriously decreased production of hemoglobin (Hb). In the majority of cases, thalassemia intermedia concerns β-globin gene pathology. The molecular basis of thalassemia intermedia is heterogeneous. Here we describe a case of an adopted child native of Myanmar suffering from β-thalassemia intermedia which was proved to be secondary to a β-zero thalassemia associated with a not yet described deletional form of HPFH. Patient, Material and Methods Male child born in 1994 with Hb varying between 50 and 60 g/l, with Hb A2 of 2.1% and Hb F of 97.9%. No α-thalassemia or α-gene triplication was found. Sequencing of β-globin gene put in evidence the IVS-I-1 (G>T) or c.92+1G>T mutation in a “homozygous” state. This mutation is known to produce a β-zero thalassemia. The patient was treated with hydroxyurea as well as with erythropoietin and the Hb value was improved up to 86 g/l with normal leucocytes and platelets count. No transfusion was given during this period of treatment. Because the clinical phenotype was not typical for β-thalassemia major homozygous for the above mentioned mutation, we analyzed β-globin cluster looking for the presence of a possible deletion responsible for Hb F activation. Patient’s DNA was extracted with commercial columns from peripheral blood cells. Analysis of deletion in the beta cluster was performed by MLPA (Multiplex Ligation Probe Analysis) MRC-Holland P-102 probe mix. The data obtained were analyzed with the Coffyanalyzer software. The exact size of the deletion was determined by PCR with the primers: DelHBB_F: 5’-AGGCTTGGCTCCTGTTTAGT-3’, DelHBB_R: 5’-TGAGAG CTGCTGAGTTGTGT-3’ Results A heterozygous deletion in the beta-globin cluster has been detected by MLPA. This deletion was located between the coordinated 5,237,089 and 5,251,133 on chromosome 11 - (GRCh37/hg19 Assembly). The deletion starts about 0.5 kb 5’ upstream the HBB gene, between HBB and HBD genes, and ends about 9 kb downstream the 3’ end of HBB gene. The density of the MLPA probes is not sufficient to determinate the exact size of the deletion (between 14.3kb and 9.6 kb). A PCR using the primers DelHBB_F and DelHBB_R determined the size of this deletion to around 11kb. Conclusions Our molecular biology studies confirmed our clinical suspicion of association of HPFH with β-zero thalassemia. In fact, we put in evidence a not yet described (to our knowledge) 11kb deletion, which is very similar to the 12.6kb deletion of the Dutch β-zero thalassemia (Br J Haematol 67:369;1987) and to the Asian Indian 10.3kb deletion described by Craig et al (Br J Haematol 82:735;1992). Our deletion starts between δ and β-globin gene, almost 0.5 kb upstream of the β-gene, and goes about 9 kb downstream of 3’ end of the β-gene. The exact borders of the deletion are currently under investigation by PCR and appropriate primers. The pathophysiology of reactivation of γ-globin genes in our case is not yet known. We raise the following hypothesis: does this deletion bring an enhancer located 3’ to β-globin gene, close enough to the γ-genes, so that transcription of these genes continues after birth? In vitro studies in expression systems (constructs) are currently performed to elucidate the exact mechanism of γ-globin activation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro.

1988 ◽  
Vol 8 (2) ◽  
pp. 860-866 ◽  
Author(s):  
P J Furdon ◽  
R Kole
Keyword(s):  
Splice Site ◽  
Mrna Splicing ◽  
Beta Globin ◽  
Specific Sequence ◽  
In Vitro Splicing ◽  
Intron 2 ◽  
Specific Sequences

We have shown previously that truncation of the human beta-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit beta-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.

scholarly journals Effects of exon sequences on splicing of model pre-mRNA substrates in vitro.

1996 ◽  
Vol 43 (1) ◽  
pp. 161-173 ◽  
Author(s):  
Z Dominski ◽  
R Kole
Keyword(s):  
Globin Gene ◽  
Beta Globin ◽  
Splice Site Selection ◽  
Sequence Composition ◽  
Recognition Element ◽  
Internal Exon ◽  
Alternatively Spliced ◽  
In Vitro Splicing

We used several related pre-mRNA substrates consisting of two introns and three exons to study effects of exon sequences on in vitro splicing. By varying the sequence of the internal exon and measuring the frequency of its skipping we confirmed that 26-nucleotide exon element naturally existing in beta-globin gene and previously analysed in vivo, has a strong stimulatory effect on splicing. Sequence analysis of this element suggests that it belongs to a family of purine-rich splicing elements found in exons of several alternatively spliced pre-mRNAs. The 26-nucleotide element can efficiently function in enhancing inclusion of internal exons regardless of their size and sequence composition, suggesting that it plays a role of a general exon recognition element. The purine-rich element is dispensable in exons flanked by strong splice sites, which promote efficient inclusion of otherwise poorly recognized exons. A row of six cytidines inserted into the internal exon (GC2 mutation) initially considered to stimulate exon inclusion to a similar extent as the purine-rich element (Dominski & Kole, 1994, J. Biol. Chem. 269, 23590-23596), appears not to affect splice site selection in vitro, and in vivo it is likely to act by stabilizing mRNA that includes the internal exon against rapid cytoplasmic degradation.

Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes

1987 ◽  
Vol 7 (1) ◽  
pp. 281-293
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Branch Site ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Branch Formation ◽  
Splice Site Sequence ◽  
Multiple Interactions ◽  
Small Nuclear Ribonucleoproteins

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

scholarly journals Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes.

1987 ◽  
Vol 7 (1) ◽  
pp. 281-293 ◽  
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Branch Site ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Branch Formation ◽  
Splice Site Sequence ◽  
Multiple Interactions ◽  
Small Nuclear Ribonucleoproteins

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

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

Abstract 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.

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