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PubMed1994

Genetic evidence for IS1 transposition regulated by InsA and the delta InsA-B'-InsB species, which is generated by translation from two alternative internal initiation sites and frameshifting.

Insertion sequence IS1 contains two reading frames, insA and B'-insB, which are responsible for its transposition, and was previously shown to express two proteins. The first, InsA, is the product of insA. The second, InsA-B'-InsB is a fusion of InsA with the product of B'-insB. Synthesis of this protein occurs by a -1 frameshift from the 3' region of the insA frame to the open reading frame B', extending from the 5' end of the insB frame. Here, I have shown genetically that IS1 encodes the third species delta InsA-B'-InsB: delta InsA-B'-InsB uses two alternative initiation codons in the middle of the insA frame, and is produced by a frameshift mechanism similar to that used in InsA-B'-InsB expression. Deletion of the small region preceding these initiation codons resulted in decreased expression of delta InsA-B'-InsB, suggesting that the small region play some role in the translation initiation. Surprisingly, it was found that delta InsA-B'-InsB has a transposase-like function and InsA can stimulate the transposition promoted by delta InsA-B'-InsB, while delta InsA-B'-InsB seemed to bind to the left terminal inverted repeat (IRL) of IS1 and inhibit transposition when it was present in excess, as well as InsA represses transposition. It is likely that IS1 transposition activity depends on the ratio of InsA to delta InsA-B'-InsB. A double missense mutation of the internal initiation codons resulted in decreased cointegration activity, showing that delta InsA-B'-InsB is responsible for transposition but InsA-B'-InsB is probably not. Some IS elements, which also contain two tandem, out-of-phase, overlapping genes, appear to express deleted fusion proteins like delta InsA-B'-InsB, but the functions are unknown. The complex phenomena of transposition and its control found in IS1 may be more general in the other mobile DNAs.

Amino Acid Sequence↗

PubMed1992

HbH disease in Sardinia: molecular, hematological and clinical aspects.

In this study we have defined the molecular basis and correlated the clinical phenotype with the alpha-globin genotype in a large series of patients of Sardinian descent with HbH disease. The most prevalent molecular defect was the deletion of 3 alpha-globin structural genes most commonly the (--/-alpha 3.7) genotype (83.6%) and rarely the (--/-alpha 4.2) genotype (1.4%), followed in decreasing order of incidence by the combination of deletion alpha zero-thalassemia and initiation codon mutation of the alpha 2-gene (--/alpha NcoI alpha = 9.8%), deletion alpha zero-thalassemia and pentanucleotide deletion of IVS-I of the alpha 2-globin gene, (--/alpha HphI alpha = 3.3%) deletion alpha zero-thalassemia and initiation codon mutation of the alpha 1-gene (--/alpha alpha NcoI = 1.3%), a homozygous state for initiation codon mutation of the alpha 2-gene (alpha Nco alpha/alpha NcoI alpha = 0.7%). Patients with the (--/alpha thal alpha) genotypes showed severer clinical and hematological features as compared to those with the (--/-alpha) or those with the (--/alpha alpha thal) genotypes. The single patient with the (alpha Nco alpha/alpha Nco alpha) genotype had a clinical phenotype intermediate between HbH disease and the alpha-thalassemia carrier status. This heterogeneity depends on the fact that the alpha 2-globin gene produces 2-3 times alpha-globin chains than the alpha 1-gene and the single remaining alpha 1-like globin gene in the -alpha 3.7 chromosome has a compensatory increase in the alpha-globin chain output. alpha-Globin gene mapping of HbH disease patients may be useful for predicting the clinical outcome and to improve genetic counseling.

Adult↗

PubMed1989

Nucleotide sequence analysis of the purEK operon encoding 5'-phosphoribosyl-5-aminoimidazole carboxylase of Escherichia coli K-12.

5'-Phosphoribosyl-5-aminoimidazole (AIR) carboxylase (EC 4.1.1.21) catalyzes step 6, the carboxylation of AIR to 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid, in the de novo biosynthesis of purine nucleotides. As deduced from the DNA sequence of restriction fragments encoding AIR carboxylase and supported by maxicell analyses, AIR carboxylase was found to be composed of two nonidentical subunits. In agreement with established complementation data, the catalytic subunit (deduced Mr, 17,782) was encoded by the purE gene, while the CO2-binding subunit (deduced Mr, 39,385) was encoded by the purK gene. These two genes formed an operon in which the termination codon of the purE gene overlapped the initiation codon of the purK gene. The 5' end of the purEK mRNA was determined by mung bean nuclease mapping and was located 41 nucleotides upstream of the proposed initiation codon. The purEK operon is regulated by the purR gene product, and a purR regulatory-protein-binding site related to the sequences found in other pur loci was identified in the purEK operon control region.

Amino Acid Sequence↗

PubMed1995

Major histocompatibility class I molecules can present cryptic translation products to T-cells.

Self or foreign cellular proteins provide peptides for presentation by major histocompatibility complex (MHC) class I molecules on the surface of antigen presenting cells (APC). Surprisingly, several studies have shown that T-cells can recognize APC transfected with antigen genes that were not present in the appropriate translational context. To understand the basis of this phenomenon, APC were transfected with DNA constructs encoding the OVA257-264 (SL8) peptide, but with varying translation initiation codons. We report that, in addition to ATG, 6 other codons (ATT, ACG, CTG, GCG, TGG, GAT) also allowed presentation to SL8-Kb-specific T-cells. Significantly, this set includes 3 of 4 known non-ATG translation initiation codons strongly suggesting that cryptic translation accounts for this phenomenon. Although expression of the SL8-Kb complex was readily detected by T-cell activation, the amount of processed peptides was below detection limit (< 30 copies/cell) in cell extracts. Thus, the fortuitous presence of these cryptic translation initiation sites in transcribed genes can explain how peptide MHC complexes were obtained in sufficient amounts for T-cell activation. The translation initiation codons identified here could also be useful for identifying potential open reading frames that possess biological and/or immunological activities.

Amino Acid Sequence↗

PubMed1999

Bronchial smooth muscle hypoplasia in mouse embryonic lungs exposed to a laminin beta1 chain antisense oligonucleotide.

We used an antisense oligonucleotide (ODN) to inhibit laminin (LM) beta1 chain synthesis in mouse embryonic lung explants and cell cultures. The ODN spanned 17 bases located 13 bases downstream the initiation codon and contained phosphorothioate and C-5 propynyl pyrimidine modifications. Penetration of the ODN into the lung explants was confirmed by fluorescein isothiocyanate (FITC) tagging. 50 microM of antisense ODN decreased LM beta1 chain synthesis by 82+/-6.9% with no significant changes in the synthesis of other LM chains. The same antisense probe but without C-5 propynyl pyrimidine modification, another 17-mer ODN complementary to the LM beta1 initiation codon, and a 17-mer ODN complementary to the LM alpha1 initiation codon had no antisense activity. Lung explants exposed to the active LM beta1 antisense ODN showed decreased LM-1 and collagen type IV deposition at the epithelial-mesenchymal interface and an arrest in bronchial smooth muscle (SM) development. Histological examination and cell motility assays suggested that this arrest was due to impaired spreading and migration of SM cell precursors over the defective basement membrane (BM). Our studies indicate that beta1-chain containing LMs play a role in bronchial myogenesis.

Animals↗

PubMed1999

Antisense oligonucleotide inhibition of hepatitis C virus (HCV) gene expression in livers of mice infected with an HCV-vaccinia virus recombinant.

Hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis worldwide. Current treatments are not curative for most infected individuals, and there is an urgent need for both novel therapeutic agents and small-animal models which can be used to evaluate candidate drugs. A small-animal model of HCV gene expression was developed with recombinant vaccinia virus vectors. VHCV-IRES (internal ribosome entry site) is a recombinant vaccinia viral vector containing the HCV 5' nontranslated region (5'-NTR) and a portion of the HCV core coding region fused to the firefly luciferase gene. Intraperitoneal injection of VHCV-IRES produced high levels of luciferase activity in the livers of BALB/c mice. Antisense oligonucleotides complementary to the HCV 5'-NTR and translation initiation codon regions were then evaluated for their effects on the expression of these target HCV sequences in BALB/c mice infected with the vaccinia virus vector. Treatment of VHCV-IRES-infected mice with 20-base phosphorothioate oligonucleotides complementary to the sequence surrounding the HCV initiation codon (nucleotides 330 to 349) specifically reduced luciferase expression in the livers in a dose-dependent manner. Inhibition of HCV reporter gene expression in this small-animal model suggests that antisense oligonucleotides may provide a novel therapy for treatment of chronic HCV infection.

Animals↗

PubMed1991

Occurrence of chloroplast ribosome recognition sites within conserved elements of the RNA genomes of carlaviruses.

The nucleotide sequences upstream from the carlavirus open reading frames were examined for direct sequence homology. Blocks of homology were evident upstream from the 25 K ORFs of potato virus S (PVS), potato virus M (PVM) and lily symptomless virus (LSV), and upstream from the coat protein initiation codons of PVS, PVM, LSV, carnation latent virus and Helenium virus S. These blocks, which correspond to the 5'-terminal regions of the subgenomic RNAs, were shown to contain potential ribosome recognition sequences. The distances between the binding sites and initiation codons ranged from 20 to 40 nucleotides on the viral RNAs. Whilst the majority of chloroplasts mRNAs have a distance of 8 nucleotides between binding site and initiation codon, the remaining have a distance of 23 nucleotides which is similar to that reported here for the carlaviruses.

Base Sequence↗

PubMed2005

The evolution of transcription-initiation sites.

Unlike the situation in prokaryotes, most eukaryotic messenger RNAs contain a moderately long 5' untranslated region (UTR). Such leader sequences impose a burden on eukaryotic genes by providing substrate for the mutational origin of premature translation-initiation codons, which generally result in defective proteins. To gain an insight into the expansion of 5' UTRs in eukaryotic genomes, we present a simple null model in which the evolution of transcription-initiation sites is entirely driven by the stochastic mutational flux of core-promoter sequences and premature translation-initiation codons. This model yields results consistent with a variety of heretofore disconnected observations, including the form of length distributions of 5' UTRs, the relatively low variance in UTR features among distantly related eukaryotes, the universal reliance on relatively simple core-promoter sequences, and the elevated density of introns in the 5' UTR. We suggest that the reduced effective population sizes of most eukaryotes impose a population-genetic environment conducive to the movement of core promoters to random positions, subject to the constraint imposed by the upstream accumulation of premature translation-initiation codons. If this hypothesis is correct, then selection for gene-specific regulatory features need not be invoked to explain either the origin of lengthy eukaryotic 5' UTRs or the 1,000-fold range of 5'-UTR lengths among genes within species. Nevertheless, once permanently established, expanded 5' UTRs may have provided a novel substrate for the evolution of mechanisms for posttranscriptional regulation of eukaryotic gene expression. These results provide a further example of how an increase in the power of random genetic drift can passively promote the evolution of forms of gene architecture that ultimately facilitate the evolution of organismal complexity.

5' Untranslated Regions↗

PubMed1989

Studies of c-myb gene regulation in MRL-lpr/lpr mice. Identification of a 5' c-myb nuclear protein binding site and high levels of binding factors in nuclear extracts of lpr/lpr lymph node cells.

Lymph node T cells of MRL-lpr/lpr mice are characterized by the production of very large amounts of c-myb mRNA. To study the control of c-myb expression, a search was made for sites on the 5' c-myb gene which could bind regulatory proteins. DNase I digestion of nuclear chromatin uncovered four DNase I hypersensitive sites in the first intron of the c-myb gene, and a single site approximately 300 bp 5' to the initiation codon. Lambda exonuclease digestion of a 5'-myb fragment in the presence of nuclear extracts from either MRL-lpr/lpr PLN or EL-4 thymoma revealed stop sites approximately 300 bp 5' (-271 to -322) to the ATG initiation codon. DNase I footprint analysis demonstrated a guanine-cytosine enriched region of potential binding sites (-274 to -319) in the region of the stop sites and a fifth potential binding site closer to the initiation codon (-163 to -168). Specific gel shift bands were detected by a 5'-myb fragment (-346 to -155) with extracts from a number of different lymphoid cell lines and the appropriate specific and non-specific competitor DNA. The DNA giving rise to these gel shift bands encompassed the region defined by the stop site and footprinting studies. To determine whether or not the protein binding to the 5' c-myb gene at -274 to -319 was associated with increased c-myb mRNA, we studied nuclear extracts of several cell lines and compared the amount of binding to the amount of c-myb mRNA found on Northern analyses. Among the cell lines, there was a correlation between c-myb expression and the amount of the 5'-myb DNA binding protein. In addition, MRL-lpr/lpr lymph node cells had high c-myb expression and large amounts of the 5'-myb binding protein. This result suggests that the binding may play some role in the c-myb expression. Moreover, the most immature cell lines had the greatest amount of the binding factor, suggesting that its regulatory effect on c-myb expression might be important in early differentiation events.

Animals↗

PubMed1990

Target dependence of antisense oligodeoxynucleotide inhibition of c-Ha-ras p21 expression and focus formation in T24-transformed NIH3T3 cells.

The relationship of antisense oligodeoxynucleotide inhibition to the predicted secondary structure of human c-Ha-ras oncogene mRNA was examined. Three antisense pentadecade-oxynucleotides complementary to the 5' cap region, a predicted loop in the 5' untranslated region, and the initiation codon region of c-Ha-ras mRNA were synthesized. T24-transformed NIH3T3 cells were treated for 24 hr with each anti-ras oligomer or control sequence. The levels of c-Ha-ras p21 antigen were then analyzed by radioimmunoprecipitation. Inhibition of p21 expression was sequence-specific and dose-dependent. The efficacy of the cap sequence in reducing p21 expression was greater than that of the initiation codon target, which in turn was more effective than the target upstream of the initiation codon. Antisense inhibition of p21 expression correlated with inhibition of focus formation.

Animals↗

PubMed2000

Inefficient function of the signal sequence of PTHrP for targeting into the secretory pathway.

Parathyroid hormone-related peptide (PTHrP) is not only secreted out of cells, but also targeted to the nucleoli due to a nucleolar targeting signal (NTS). We assessed the molecular mechanism underlying the dual targeting of PTHrP by constructing a series of truncated forms of rat PTHrP cDNA and expressing them in CHO cells. Immunostaining was observed in both the Golgi apparatus and nucleoli in the same cell expressing PTHrP with the N-terminal full-length signal sequence. When PTHrP molecules were translated from CUGs downstream of the AUG-initiator codon in the signal sequences, potential alternative initiators of the translation, they were exclusively localized in the nucleoli. In contrast, when a construct containing only the ATG-initiator codon was expressed, PTHrP was found to localize in both the nucleolus and the Golgi apparatus. No nucleolar staining of PTHrP was observed in the CHO cells transfected with PTH/PTHrP receptors even after incubating with a conditioned medium containing PTHrP, ruling out a possibility that PTHrP is, once secreted, internalized via receptor-mediated endocytosis and subsequently conveyed to nucleoli. Compatible with these morphological observations, a preproform of PTHrP was found in the cells expressing PTHrP in addition to proPTHrP, indicative of molecules along the secretory pathway. These results strongly indicate that the signal sequence of PTHrP is not sufficient to direct all the newly synthesized molecules across the endoplasmic reticulum, resulting in part of it being delivered to the nucleoli due to the NTS.

Amino Acid Sequence↗

PubMed1985

Effect of dinucleotides on wheat germ translation system.

The effect of ribodinucleoside monophosphates on total protein synthesis was studied in a wheat germ cell-free system, using brome mosaic virus (BMV) RNA as a messenger. Dinucleotides inhibit total protein synthesis to different extents. Of those tested the most inhibitory is CpA. The inhibitory effect of dinucleotides is due to their adverse effect on initiation and not on elongation of polypeptide synthesis. It seems that the dinucleotides complementary to the initiation codon are able to compete with the initiator tRNA during initiation of protein synthesis. The comparison of the effect exerted by different dinucleotides suggests that under conditions of the in vitro protein synthesis RNA 4 is an mRNA molecule with the initiation codon and its immediate neighbourhood being exposed.

Cell-Free System↗

PubMed1991

Mistargeting of peroxisomal L-alanine:glyoxylate aminotransferase to mitochondria in primary hyperoxaluria patients depends upon activation of a cryptic mitochondrial targeting sequence by a point mutation.

In approximately one-third of primary hyperoxaluria type 1 patients, disease is associated with a unique protein sorting defect in which hepatic L-alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44), which is normally peroxisomal, is mistargeted to mitochondria. In all such patients analyzed to date, the gene encoding the aberrantly targeted AGT carries three point mutations, each of which specifies an amino acid substitution. In this paper we show that one of these substitutions, a proline-to-leucine at residue 11, is necessary and sufficient for the generation of a mitochondrial targeting sequence in the AGT protein. AGT with this substitution appears to interact specifically with the mitochondrial protein import machinery, via a discrete N-terminal domain of the AGT protein. The N-terminal 19 amino acids of AGT with this substitution are sufficient to direct mouse cytosolic dihydrofolate reductase to mitochondria, and a synthetic peptide corresponding to this same 19-amino acid region reversibly inhibits mitochondrial protein import, not only of AGT but also of ornithine transcarbamoylase, a genuine cytoplasmically synthesized mitochondrial protein. We have extended these studies to analyze a region of normal human AGT cDNA directly upstream of the coding region. This sequence appears to correspond to an ancestral mitochondrial targeting sequence deleted from the human coding region by point mutation at the initiation codon. We show that reestablishment of this initiation codon produces an active mitochondrial targeting sequence that is different to that found in the hyperoxaluria patients. These results are discussed with reference to the AGT targeting defect in primary hyperoxaluria and also in relation to the highly unusual species specificity of subcellular distribution of AGT among mammals.

Alanine Transaminase↗

PubMed2000

Inactivation Analysis of HcNPV Cysteine Protease Gene and Chitinase Gene.

The fragment of Hyphantria cunea nuclear polyhedrosis virus (HcNPV), which contains cysteine protease gene(CP) and chitinase gene (ChiA),was inserted into plasmid PCRII to construct transfer vector pHcCVdel. Recombinant transfer vector pHcCvpolh was constructed by inserting the fragment of HcNPV polyhedrin gene (polh) into the EcoRI site of the pHcCVdel. By contransfection of the pHcCvpolh and HcNPV-PTTH(+) DNA, which contained Bombyx mori prothoracicotropic hormone gene (PTTH) into SPIM cells, the region from +76 downstream of ChiA gene initiation codon to +20 downstream of CP gene initiation codon was substituted by the polh gene, and the recombinant virus HcNPVpolh(+)CP(-)ChiA(-)PTTH(+) was produced. The recombinant virus, in which CP gene and ChiA gene were inactive, could express PTTH gene and form polyhedra. SPIM cells infected with the recombinant virus revealed that CP and ChiA gene were not essential for viral replication and had no significant effect on polyhedron formation, but the infected cells survived 2 days more than those infected with HcNPV and HcNPVPTTH(+). Therefore, the expression time of foreign gene may be prolonged when CP gene and ChiA gene were inactivated.

Journal Article↗

PubMed1990

Expression of human immunodeficiency virus 1 (HIV-1) envelope gene products transcribed from a heterologous promoter. Kinetics of HIV-1 envelope processing in transfected cells.

The expression of human immunodeficiency virus 1 (HIV-1) envelope glycoprotein products was studied in cells transfected with env gene constructs transcribed from an SV40 promoter. Gene constructs possessing the complete tat, rev (tat+ rev+) and env genes were transiently expressed in COS-1 cells as precursor SU-TM (gp160), SU.TM (gp120 x 41), and nucleolar rev protein. In addition, envelope glycoprotein was detected on the surface of those transfected COS-1 cells expressing abundant levels of env protein. Transfected constructs possessing a mutated tat translational initiation codon (tat-rev+) were expressed in COS-1 cells with at least a 10-fold increase in the level of envelope glycoprotein expression compared to the analogous constructs with an intact tat AUG codon (tat+ rev+). Mutation of the rev initiation codon (tat+ rev-) and (tat-rev-) resulted in no detectable expression of env products but expression of these proteins could be rescued by co-transfection of a cDNA encoding the rev gene. Subgenomic tat/rev transcripts were detected following transfection of all of the gene constructs indicating splicing of the env mRNAs transcribed from a heterologous promoter. Unspliced env transcripts were only detected in the cytoplasm of cells transfected with (rev+) constructs or with the (tat- rev-) construct in the presence of the rev cDNA supplied in trans. In contrast, unspliced transcripts were detected in the nuclear and total cellular RNA of all transfected cells. Expression of rev protein was localized to the nucleolus of transfected COS-1 cells. These results indicate that the export of unspliced env mRNA to the cytoplasm is facilitated by the expression of rev. Following env synthesis, the conversion of SU-TM (gp160) to SU.TM (gp120 x 41) was not quantitative. After a 20-h pulse-chase, only 40% of the SU-TM (gp160) present at the start of the chase period was subsequently accountable as mature SU (gp120), and approximately 30% of the detectable SU (gp120) was found in the culture medium of transfected COS-1 cells. The findings indicate that the surface expression of SU.TM (gp120 x 41) derived from heterologous gene transcripts is modulated (i) the co-expression of rev, (ii) the efficiency of proteolytic processing of SU-TM (gp160), and (iii) the degree of SU (gp120) shedding and/or secretion from the cell.

Animals↗

PubMed2000

RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1.

The chloroplast ribosomal protein CS1, the homolog of the bacterial ribosomal protein S1, is believed to be involved in the process of ribosome binding to mRNA during translation. Since translation control is an important step in chloroplast gene expression, and in order to study initiation complex formation, we studied the RNA-binding properties of CS1 protein. We found that most of the CS1 protein in spinach chloroplast co-purified with the 30S ribosomal subunit. The relative binding affinity of RNA to CS1 was determined using the UV-crosslinking competition assay. CS1 protein binds the ribohomopolymer poly(U) with a relatively high binding affinity. Very low binding affinities were obtained for the other ribohomopolymers, poly(G), poly(A) and poly(C). In addition, no specific binding of CS1, either in the 30S complex or as a recombinant purified protein, was obtained to the 5'-untranslated region of the mRNA in comparison to the other parts. RNA-binding experiments, in which the N- and C-termini of the protein were analyzed, revealed that the RNA-binding site is located in the C-terminus half of the protein. These results suggest that CS1 does not direct the 30S complex to the initiation codon of the translation site by specific binding to the 5'-untranslated region. In bacteria, specific binding is derived by base pairing between 16S rRNA and the Shine-Dalagarno sequences. In the chloroplast, nuclear encoded and gene-specific translation factors may be involved in the determination of specific binding of the 30S subunit to the initiator codon.

5' Untranslated Regions↗

PubMed2002

The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes.

Classical swine fever virus (CSFV) is a member of the pestivirus family, which shares many features in common with hepatitis C virus (HCV). It is shown here that CSFV has an exceptionally efficient cis-acting internal ribosome entry segment (IRES), which, like that of HCV, is strongly influenced by the sequences immediately downstream of the initiation codon, and is optimal with viral coding sequences in this position. Constructs that retained 17 or more codons of viral coding sequence exhibited full IRES activity, but with only 12 codons, activity was approximately 66% of maximum in vitro (though close to maximum in transfected BHK cells), whereas with just 3 codons or fewer, the activity was only approximately 15% of maximum. The minimal coding region elements required for high activity were exchanged between HCV and CSFV. Although maximum activity was observed in each case with the homologous combination of coding region and 5' UTR, the heterologous combinations were sufficiently active to rule out a highly specific functional interplay between the 5' UTR and coding sequences. On the other hand, inversion of the coding sequences resulted in low IRES activity, particularly with the HCV coding sequences. RNA structure probing showed that the efficiency of internal initiation of these chimeric constructs correlated most closely with the degree of single-strandedness of the region around and immediately downstream of the initiation codon. The low activity IRESs could not be rescued by addition of supplementary eIF4A (the initiation factor with ATP-dependent RNA helicase activity). The extreme sensitivity to secondary structure around the initiation codon is likely to be due to the fact that the eIF4F complex (which has eIF4A as one of its subunits) is not required for and does not participate in initiation on these IRESs.

5' Untranslated Regions↗

PubMed1986

Molecular pathology of haemoglobin H disease in Sardinians.

We investigated the molecular basis for haemoglobin H disease in 50 Sardinian patients by restriction endonuclease analysis. We found that the majority (78% of the cases) are due to gene deletion (- -/- alpha). Among those with a combination of deletion and nondeletion defects (- -/alpha alpha th), the most prevalent nondeletion lesion (70% of the nondeletion defects) was the initiation codon mutation of the alpha 2 gene (alpha Nco alpha), previously discovered in this population. Of the remaining patients with the (- -/alpha alpha th) genotype, two showed the IVS-1 splice junction lesion and one a mutation in the alpha 1 gene, removing the Nco I site within the 5' part of the alpha 1 gene, which may arise from a process of gene conversion from the initiation codon mutant of the alpha 2 gene. A single patient had the homozygous state for the initiation codon mutant of the alpha 2 gene. Study of genotype-phenotype correlations indicates that the (alpha Nco alpha) haplotype is associated with a more severe defect in the alpha-globin chain output than that resulting from the (-alpha) haplotype. We may conclude that restriction endonuclease analysis is a powerful method for the definition of the molecular heterogeneity of haemoglobin H disease.

Adolescent↗