S. E. Antonarakis

Molecular characterization of seven beta-thalassemia mutations in Asian Indians.

To characterize systematically the mutations which produce beta‐thalassemia in Asian Indians, we first determined the DNA polymorphism haplotype in the beta‐globin gene cluster of 44 beta‐thalassemia chromosomes in the ethnic group. Nine different haplotypes were observed. Upon molecular cloning and partial DNA sequencing of one beta‐gene from each of eight haplotypes and two from the ninth, seven different mutations were found. None of these have been identified in Mediterranean patients, even among the five haplotypes which appeared identical in the two groups. Asian Indian mutations included one nonsense and three frameshift mutations, one deletion affecting an acceptor splice site, and two mutations affecting a donor splice site. The correlation of a specific mutation with a specific haplotype was high but not invariant. Two mutations were associated with more than one haplotype but, in each instance, the mutation spread to a new haplotype could be explained most simply by recombination 5′ to the beta‐globin gene. In addition, four mutations, one reported here and three others previously reported, have been observed on two chromosome backgrounds that are identical except for the status of a polymorphic HinfI site 5′ to the beta gene. This HinfI site does not show significant linkage disequilibrium with markers both 5′ and 3′ to it, suggesting that it lies within a region of relative sequence randomization.

Thalassemia due to a mutation in the cleavage-polyadenylation signal of the human beta-globin gene.

A beta‐globin gene cloned from a person with beta‐thalassemia contained a T‐‐‐‐C substitution within the conserved sequence AATAAA that forms a portion of the recognition signal for endonucleolytic cleavage and polyadenylation of primary mRNA transcripts. By Northern blot analysis a novel beta‐globin RNA species, 1500 nucleotides in length, was detected in erythroid RNA. Nuclease protection studies of erythroid RNA, as well as RNA generated upon transient expression of the cloned mutant gene in HeLa cells, located the 3′ terminus of this novel, polyadenylated RNA 900 nucleotides downstream of the normal poly(A) addition site, within 15 nucleotides of the first AATAAA in the 3′‐flanking region of the beta‐globin gene. These findings define the in vivo terminus of an elongated RNA and establish that human beta‐globin transcription may extend at least 900 nucleotides 3′ of the normal polyadenylation site.

New class of gene-termini-associated human RNAs suggests a novel RNA copying mechanism

Small (<200 nucleotide) RNA (sRNA) profiling of human cells using various technologies demonstrates unexpected complexity of sRNAs with hundreds of thousands of sRNA species present. Genetic and in vitro studies show that these RNAs are not merely degradation products of longer transcripts but could indeed have a function. Furthermore, profiling of RNAs, including the sRNAs, can reveal not only novel transcripts, but also make clear predictions about the existence and properties of novel biochemical pathways operating in a cell. For example, sRNA profiling in human cells indicated the existence of an unknown capping mechanism operating on cleaved RNA, a biochemical component of which was later identified. Here we show that human cells contain a novel type of sRNA that has non-genomically encoded 5′ poly(U) tails. The presence of these RNAs at the termini of genes, specifically at the very 3′ ends of known mRNAs, strongly argues for the presence of a yet uncharacterized endogenous biochemical pathway in cells that can copy RNA. We show that this pathway can operate on multiple genes, with specific enrichment towards transcript-encoding components of the translational machinery. Finally, we show that genes are also flanked by sense, 3′ polyadenylated sRNAs that are likely to be capped.

A linkage map of three anonymous human DNA fragments and SOD-1 on chromosome 21.

Using DNA polymorphisms adjacent to single‐copy genomic fragments derived from human chromosome 21, we initiated the construction of a linkage map of human chromosome 21. The probes were genomic EcoRI fragments pW228C, pW236B, pW231C and a portion of the superoxide dismutase gene (SOD‐1). DNA polymorphisms adjacent to each of the probes were used as markers in informative families to perform classical linkage analysis. No crossing‐over was observed between the polymorphic sites adjacent to genomic fragments pW228C and pW236B in 31 chances for recombination. Therefore, these fragments are closely linked to one another (theta = 0.00, lod score = 6.91, 95% confidence limits = 0‐10 cM) and can be treated as one ‘locus’ with four high‐frequency markers. There is a high degree of non‐random association of markers adjacent to each of these two probes which suggests that they are physically very close to one another in the genome. The pW228C ‐ pW236B ‘locus’ was also linked to the SOD‐1 gene (theta = 0.07, lod score = 4.33, 95% confidence limits = 1‐20 cM). On the other hand, no evidence for linkage was found between the pW228C‐pW236B ‘locus’ and the genomic fragment pW231C (theta = 0.5, lod score = 0.00). Based on the fact that pW231C maps to 21q22.3 and SOD‐1 to 21q22.1, we suggest that the pW228C‐pW236B ‘locus’ lies in the proximal long arm of chromosome 21. These data provide the outline of a linkage map for the long arm of chromosome 21, and indicate that the pW228C‐pW236B ‘locus’ is a useful marker system to differentiate various chromosome 21s in a population.

Linkage mapping of the AML1 gene on human chromosome 21 using a DNA polymorphism in the 3′ untranslated region

We have detected a polymorphism in the 3 untranslated region of the AML1 gene, which is located at the breakpoint on chromosome 21 in the t(8;21)(q22;q22.3) translocation often associated with patients with acute myeloid leukemia. Informative CEPH families were genotyped for this polymorphism and used to localize the gene on the linkage map of human chromosome 21. The AML1 gene is located between the markers D21S216 and D21S211, in chromosomal band 21q22.3.