The two intervening sequences of human beta- and gamma-globin pre-mRNAs are excised in a preferred temporal order in vitro.

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3428 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3428-3437 ◽

Cited By ~ 15

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.

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Point mutation associated with hereditary persistence of fetal hemoglobin decreases RNA polymerase III transcription upstream of the affected gamma-globin gene

Molecular and Cellular Biology ◽

10.1128/mcb.6.9.3278-3282.1986 ◽

1986 ◽

Vol 6 (9) ◽

pp. 3278-3282

Author(s):

D P Carlson ◽

J Ross

Keyword(s):

Rna Polymerase ◽

Globin Gene ◽

Rna Polymerase Iii ◽

Fetal Hemoglobin ◽

In Vitro Transcription ◽

Base Substitution ◽

Gamma Globin ◽

Polymerase Iii ◽

Hemoglobin Production

A base substitution in the 5'-flanking region of a human fetal globin gene is associated with abnormal fetal hemoglobin production. It also reduces by 5- to 10-fold in vitro transcription of the gene by RNA polymerase III. We discuss potential links between polymerase III transcription and abnormal hemoglobin production.

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Influence of recombinant hematopoietins and of fetal bovine serum on the globin synthetic pattern of human BFUe

Blood ◽

10.1182/blood.v76.6.1150.1150 ◽

1990 ◽

Vol 76 (6) ◽

pp. 1150-1157 ◽

Cited By ~ 21

Author(s):

AR Migliaccio ◽

G Migliaccio ◽

M Brice ◽

P Constantoulakis ◽

G Stamatoyannopoulos ◽

...

Keyword(s):

Bovine Serum ◽

Fetal Bovine Serum ◽

Mrna Level ◽

Fetal Hemoglobin ◽

Mrna Levels ◽

Gm Csf ◽

Gamma Globin ◽

Fetal Bovine ◽

Globin Synthesis

Abstract We have studied the effects of recombinant hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-3 (IL-3) on the globin program of adult human erythroid progenitors (BFUe) stimulated to terminal differentiation by erythropoietin under fetal bovine serum (FBS)-supplemented or FBS- deprived culture conditions. Fetal globin production by BFUe-derived erythroblasts was assessed at the protein and mRNA level and its cellular distribution was evaluated by immunofluorescence. Although hemoglobinization and maturation of BFUe-derived erythroblasts was by and large comparable in FBS-replete versus FBS-deprived cultures, the latter had significantly less (up to 20-fold) gamma-globin and gamma- globin mRNA levels. Reduced gamma-globin in serum-deprived cultures was also reflected by a smaller proportion of erythroblasts with detectable gamma-globin by immunofluorescence. Erythroid bursts induced by either GM-CSF or IL-3 produced similar levels of gamma-globin both in FBS- supplemented and in FBS-deprived cultures. These results, obtained even in cultures of highly enriched BFUe, suggest that GM-CSF and IL-3, although they significantly increase the number and size of erythroid bursts, do not by themselves exert a direct influence on the level of fetal globin synthesis. By contrast, factor(s) present in FBS appear to exert a dominant influence on fetal globin synthesis in vitro. Although FBS-deprived conditions appear to largely abrogate the in vitro activation of fetal hemoglobin (Hb F) in normal samples, they do support increased Hb F production in samples from patients with hereditary persistence of fetal hemoglobin or from cord blood.

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On the induction of fetal hemoglobin by butyrates: in vivo and in vitro studies with sodium butyrate and comparison of combination treatments with 5-AzaC and AraC

Blood ◽

10.1182/blood.v74.6.1963.1963 ◽

1989 ◽

Vol 74 (6) ◽

pp. 1963-1971 ◽

Cited By ~ 79

Author(s):

P Constantoulakis ◽

G Knitter ◽

G Stamatoyannopoulos

Keyword(s):

Sodium Butyrate ◽

Progenitor Cell ◽

Fetal Hemoglobin ◽

Globin Genes ◽

Cell Numbers ◽

Gamma Globin ◽

Combination Treatments ◽

Bone Marrow Cultures

Abstract To obtain information on the cellular mechanism of induction of fetal hemoglobin (HbF) by sodium butyrate (NaB), we treated adult baboons with NaB and assessed its effects on HbF expression. Infusion of NaB increased F reticulocytes and F-positive CFUe and e-cluster colonies without induction of reticulocytosis or increase in progenitor cell numbers. Addition of NaB in bone marrow cultures increased the frequency of F-positive CFUe and e-clusters without increasing progenitor cell numbers. NaB induced HbF in human adult BFUe cultures and increased the gamma/gamma + beta globin chain and mRNA ratios in short-term incubations of culture-derived erythroblasts. There was a synergistic induction of HbF by NaB and 5-azacytidine (5-azaC), but not when the animal was treated with NaB and cytarabine (AraC). Our results suggest that the activation of gamma-globin expression by NaB reflects an action of this compound on globin genes or globin chromatin.

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

Blood ◽

10.1182/blood.v86.1.323.bloodjournal861323 ◽

1995 ◽

Vol 86 (1) ◽

pp. 323-328 ◽

Cited By ~ 7

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.

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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 ◽

10.1182/blood.v83.3.822.822 ◽

1994 ◽

Vol 83 (3) ◽

pp. 822-827 ◽

Cited By ~ 17

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.

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Sequence variations in the 5' flanking and IVS-II regions of the G gamma- and A gamma-globin genes of beta S chromosomes with five different haplotypes

Blood ◽

10.1182/blood.v77.11.2488.2488 ◽

1991 ◽

Vol 77 (11) ◽

pp. 2488-2496 ◽

Cited By ~ 71

Author(s):

KD Lanclos ◽

C Oner ◽

AJ Dimovski ◽

YC Gu ◽

TH Huisman

Keyword(s):

Fetal Hemoglobin ◽

Globin Genes ◽

Sequencing Data ◽

Dot Blot ◽

Hemoglobin F ◽

Gamma Globin ◽

Sequence Variations ◽

Unknown Gene ◽

Flanking Regions

Abstract We have amplified and sequenced the 5′ flanking and the second intervening sequence (IVS-II) regions of both the G gamma- and A gamma- globin genes of the beta S chromosomes from sickle cell anemia (SS) patients with homozygosities for five different haplotypes. The sequencing data, compared with previously published sequences for the normal chromosomes A and B, show many similarities to chromosome B for haplotypes 19, 20, and 17, while haplotypes 3 and 31 are remarkably similar to chromosome A and also similar to each other. Several unique mutations were found in the 5′ flanking regions (G gamma and A gamma) of haplotypes 19 and 20 and in the IVS-II segments of the same genes of haplotypes 19, 20, and 17; the IVS-II of haplotypes 3 and 31 were identical to those of chromosome A. Dot-blot analyses of amplified DNA from additional SS patients with specific probes have confirmed that these mutations are unique for each haplotype. The two general patterns that have been observed among the five haplotypes have most probably arisen by gene conversion events between the A and B type chromosomes in the African population. These patterns correlate with high and low fetal hemoglobin expression, and it is speculated that these and other yet unknown gene conversions may contribute to the variations in hemoglobin F and G gamma levels observed among SS patients. In vitro expression experiments involving the approximately 1.3-kb 5′ flanking regions of the G gamma- and A gamma-globin genes of the beta S chromosomes with the five different haplotypes failed to detect differences between the levels of expression, suggesting that the sequence variations observed between these segments of DNA are not the primary cause of the differences in hemoglobin F levels among the SS patients.

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RNase III Processing of Intervening Sequences Found in Helix 9 of 23S rRNA in the Alpha Subclass of Proteobacteria

Journal of Bacteriology ◽

10.1128/jb.182.17.4719-4729.2000 ◽

2000 ◽

Vol 182 (17) ◽

pp. 4719-4729 ◽

Cited By ~ 36

Author(s):

Elena Evguenieva-Hackenberg ◽

Gabriele Klug

Keyword(s):

Rhodobacter Capsulatus ◽

23S Rrna ◽

Recent Attempt ◽

Rnase Iii ◽

E Coli ◽

Rdna Sequences ◽

Intervening Sequences ◽

Rna Maturation

ABSTRACT We provide experimental evidence for RNase III-dependent processing in helix 9 of the 23S rRNA as a general feature of many species in the alpha subclass of Proteobacteria(alpha-Proteobacteria). We investigated 12Rhodobacter, Rhizobium,Sinorhizobium, Rhodopseudomonas, andBartonella strains. The processed region is characterized by the presence of intervening sequences (IVSs). The 23S rDNA sequences between positions 109 and 205 (Escherichia coli numbering) were determined, and potential secondary structures are proposed. Comparison of the IVSs indicates very different evolutionary rates in some phylogenetic branches, lateral genetic transfer, and evolution by insertion and/or deletion. We show that the IVS processing inRhodobacter capsulatus in vivo is RNase III-dependent and that RNase III cleaves additional sites in vitro. While all IVS-containing transcripts tested are processed in vitro by RNase III from R. capsulatus, E. coli RNase III recognizes only some of them as substrates and in these substrates frequently cleaves at different scissile bonds. These results demonstrate the different substrate specificities of the two enzymes. Although RNase III plays an important role in the rRNA, mRNA, and bacteriophage RNA maturation, its substrate specificity is still not well understood. Comparison of the IVSs of helix 9 does not hint at sequence motives involved in recognition but reveals that the “antideterminant” model, which represents the most recent attempt to explain the E. coli RNase III specificity in vitro, cannot be applied to substrates derived from alpha-Proteobacteria.

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Sequence organization of a pea chloroplast DNA gene coding for a 34,500-dalton protein.

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2556 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2556-2563 ◽

Cited By ~ 46

Author(s):

K K Oishi ◽

D R Shapiro ◽

K K Tewari

Keyword(s):

Chloroplast Dna ◽

Protein Product ◽

Sequence Organization ◽

Intervening Sequences ◽

Gene Coding ◽

Initiation Of Transcription ◽

Flanking Region ◽

Flanking Regions ◽

Vitro Protein

A gene (PGII), which codes for a 34.5-kilodalton protein, has been isolated and cloned from pea chloroplast DNA. The production of its 1.2-kilobase mRNA is photodependent. The direction of transcription has been determined, the site of initiation of transcription has been found, and an in vitro protein product has been produced. The gene, including the 5' and 3'-flanking regions, has been sequenced. It shows ca. 95% homology to the photosystem II thylakoid membrane protein, photogene 32, from spinach and tobacco. There are no intervening sequences. The 5'-flanking region suggests similarities with Escherichia coli promoters. The 5'-flanking region is remarkably conserved among pea, spinach, and tobacco DNA.

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