Structure and organization of the human ankyrin-1 gene. Basis for complexity of pre-mRNA processing.

Ankyrin-1 (ANK-1) is an erythrocyte membrane protein that is defective in many patients with hereditary spherocytosis, a common hemolytic anemia. In the red cell, ankyrin-1 provides the primary linkage between the membrane skeleton and the plasma membrane. To gain additional insight into the structure and function of this protein and to provide the necessary tools for further genetic studies of hereditary spherocytosis patients, we cloned the human ANK-1 chromosomal gene. Characterization of the ANK-1 gene genomic structure revealed that the erythroid transcript is composed of 42 exons distributed over approximately 160 kilobase pairs of DNA. Comparison of the genomic structure with the protein domains reveals a near-absolute correlation between the tandem repeats encoding the membrane-binding domain of ankyrin with the location of the intron/exon boundaries in the corresponding part of the gene. Erythroid stage-specific, complex patterns of alternative splicing were identified in the region encoding the regulatory domain of ankyrin-1. Novel brain-specific transcripts were also identified in this region, as well as in the "hinge" region between the membrane-binding and spectrin-binding domains. Utilization of alternative polyadenylation signals was found to be the basis for the previously described, stage-specific 9.0- and 7.2-kilobase pair transcripts of the ANK-1 gene.

Ankyrin-1 mutations are a major cause of dominant and recessive hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia in Northern Europeans. The primary molecular defects reside in the red blood cell (RBC) membrane, particularly in proteins that link the membrane skeleton to the overlying lipid bilayer and its integral membrane constituents. Ankyrin-1 is the predominant linker molecule. It attaches spectrin, the major skeletal protein, to the cytoplasmic domain of band 3, the RBC anion exchanger. Two-thirds of patients with HS have combined spectrin and ankyrin-1 deficiency; deficiency of band 3 occurs in about 15 to 20% (ref.1). These data suggest that ankyrin-1 or band 3 defects may be common in HS. To test this we screened all 42 coding exons plus the 5' untranslated/promoter region of ankyrin-1 and the 19 coding exons of band 3 in 46 HS families. Twelve ankyrin-1 mutations and five band 3 mutations were identified. Missense mutations and a mutation in the putative ankyrin-1 promoter were common in recessive HS. In contrast, ankyrin-1 and band 3 frameshift and nonsense null mutations prevailed in dominant HS. Increased accumulation of the normal protein product partially compensated for the ankyrin-1 or band 3 defects in some of these null mutations. Our findings indicate that ankyrin-1 mutations are a major cause of dominant and recessive HS (approximately 35 to 65%), that band 3 mutations are less common (approximately 15 to 25%), and that the severity of HS is modified by factors other than the primary gene defect.

A nonsense mutation in the erythrocyte band 3 gene associated with decreased mRNA accumulation in a kindred with dominant hereditary spherocytosis.

We studied a French kindred with typical hereditary spherocytosis (HS). Studies of erythrocytes and erythrocyte membranes from HS individuals revealed abnormal erythrocyte membrane mechanical stability as well as 15-20% deficiency of band 3, the anion transporter. Anion transport studies of red cells from two affected individuals revealed decreased sulfate flux. Nucleotide sequence of cDNA encoding the distal third of the cytoplasmic domain and the entire transmembrane domain of band 3 obtained by RT-PCR of reticulocyte RNA of an affected family member was normal. Sequence analysis of genomic DNA from an HS individual identified a nonsense mutation of the band 3 gene, Q330X, near the end of the band 3 cytoplasmic domain. This mutation was present in genomic DNA of all HS family members and absent in DNA of unaffected family members. Using an RT-PCR-based assay, a marked quantitative decrease in accumulation of the mutant band 3 RNA was detected. Thus the codon 330 nonsense mutation is responsible for the decreased accumulation of mutant band 3 RNA and the deficiency of band 3 protein in this kindred. These results have important implications for the role of band 3 defects in the membrane pathobiology of HS as well as for the techniques used in detection of HS mutations.

Developmental regulation of human gamma- and beta-globin genes in the absence of the locus control region.

Two lines of transgenic mice carrying a normal 40-kb Kpn I beta-globin cluster transgene lacking the locus control region (LCR) were analyzed for the expression of human gamma- and beta-globin genes during mouse development. After RNase protection assays, the ratios of human G gamma-, A gamma-, or beta-mRNAs relative to endogenous mouse zeta + alpha mRNAs were obtained for each stage of development. The two gamma transgenes were expressed in day-11.5 blood (embryonic stage) and day-13.5 blood (early fetal stage), but their expression was markedly decreased by day 16.5 of fetal life. Expression of the beta transgene was essentially absent at day 13.5, appeared at a low level by day 16.5, and was maximal by day 18.5, reaching a level similar to that observed in adult mice. Therefore, developmentally regulated expression of the human gamma- and beta-globin transgenes was obtained in the absence of the LCR. The relative expression of human gamma- and beta-globin genes was also examined in mice carrying 40-kb Kpn I beta-cluster transgenes with two different base substitutions associated with nondeletion forms of hereditary persistence of fetal hemoglobin (HPFH), -202 C-->G G gamma HPFH and -117 G-->A A gamma HPFH. The ratio of G gamma- to beta-globin transcripts was markedly increased in red blood cells of adult mice from three different lines carrying the transgene with the -202 G gamma HPFH mutation. This result confirms our previous preliminary results (Tanaka et al: Ann NY Acad Sci, 612:167, 1990) indicating that the -202 G gamma HPFH phenotype was reproduced in transgenic mice. The relatively low levels of G gamma-mRNA expression in adult mice carrying the non-HPFH transgene excludes a major influence of the 3' beta-globin enhancer, present upstream of the G gamma gene because of the tandem repeat insertion, as a factor in the persistent G gamma gene expression observed in blood of adult mice carrying the -202 G gamma HPFH transgene. This conclusion is also supported by the fact that, in mice carrying the -117 A gamma HPFH transgene, G gamma-globin mRNA was detected in blood of adult animals only at low levels similar to that observed in the non-HPFH lines. However, the A gamma-HPFH phenotype was not reproduced in the transgenic lines carrying the -117A gamma HPFH mice.

The exon-intron organization of the human erythroid beta-spectrin gene.

The human erythrocyte beta-spectrin gene DNA has been cloned from overlapping human genomic phage and cosmid recombinants. The entire erythroid beta-spectrin mRNA is encoded by 32 exons that range in size from 49 to 871 bases. The exon/intron junctions have been identified and the exons mapped. There is no correlation between intron positions and the repeat units of 106 amino acids within domain II of the beta-spectrin gene. The scatter of the introns over the 17 repeats argues against the 106-amino-acid unit representing a minigene that underwent repeated duplication resulting in the present beta-spectrin gene. In fact, the two largest exons, exon 14 (871 bp) and 16 (757 bp), extend over 4 and 3 repeat units of 106 amino acids, respectively, while repeat beta 10 is encoded by 4 exons. No single position of an intron in the beta-spectrin gene is conserved between any of the 17 beta-spectrin and 22 alpha-spectrin repeat units. The nucleotide sequences of the exon/intron boundaries conform to the consensus splice site sequences except for exon 20, whose 5' donor splice-site sequence begins with GC. The beta-spectrin isoform present in the human brain, the skeletal muscle, and the cardiac muscle is an alternatively spliced product of the erythroid beta-spectrin gene. This splice site is located within the coding sequences of exon 32 and its utilization in nonerythroid tissues leads to the use of 4 additional downstream exons with a size range of 44 to 530 bp.

Full-length sequence of the cDNA for human erythroid beta-spectrin.

Spectrin is the major molecular consituent of the red cell membrane skeleton. We have isolated overlapping human erythroid beta-spectrin cDNA clones and determined 6773 base pairs of contiguous nucleotide sequence. This includes the entire coding sequence of beta-spectrin. The sequence translates into a 2137 amino acid, 246-kDa peptide. beta-Spectrin is found to consist of three distinct domains. Domain I, at the N terminus, is a 272-amino acid region lacking resemblance to the spectrin repetitive motif. Sequences in this region exhibit striking sequence homology, at both nucleotide and amino acid levels, to the N-terminal "actin-binding" domains of alpha-actinin and dystrophin. Between residues 51 and 270 there is 55% amino acid identity to human dystrophin, with only four single amino acid gaps in alignment. Domain II consists of 17 spectrin repeats. Several sequence variations are observed in typical repeat structure. Homology to alpha-actinin extends beyond domain I into the N-terminal portion of domain II. Domain III, 52 amino acid residues at the C terminus, does not adhere to the spectrin repeat motif. Combining knowledge of spectrin primary structure with previously reported functional studies, it is possible to make several inferences regarding structure/function relationships within the beta-spectrin molecule.

The complete cDNA and polypeptide sequences of human erythroid alpha-spectrin.

Overlapping human erythroid alpha-spectrin cDNA clones were isolated from lambda gt11 libraries constructed from cDNAs of human fetal liver and erythroid bone marrow. The composite 8001-base pair (bp) cDNA nucleotide sequence contains 187-bp 5'- and 528-bp 3'-untranslated regions and has a single long open reading frame of 7287 bp that encodes a polypeptide of 2429 residues. As previously described (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180), spectrin is composed largely of homologous 106-amino acid repeat units. From the amino acid sequence deduced from the cDNA, alpha-spectrin can be divided into 22 segments. Segments 1-9 and 12-19 are homologous and can therefore be considered repeats; the average number of identical residues in pairwise comparisons of these repeats is 22 out of 106, or 21%. Of these 17 repeats, 11 are exactly 106 amino acids in length, whereas five others differ from this length by a single residue. Segments 11, 20, and 21, although less homologous, appear to be related to the more highly conserved repeat units. The very N-terminal 22 residues, segment 10, which is atypical both in length and sequence, and the C-terminal 150 residues in segment 22 appear to be unrelated to the conserved repeat units. The sequence of the erythroid alpha-spectrin polypeptide chain is compared to that of human alpha-fodrin and chicken alpha-actinin to which it is related. alpha-Spectrin is more distantly related to dystrophin.

Genetic and physiological characterization of ciprofloxacin resistance in Pseudomonas aeruginosa PAO.

Spontaneous ciprofloxacin-resistant mutants of Pseudomonas aeruginosa PAO2 were isolated on ML agar containing 0.5 microgram of ciprofloxacin per ml (2 times the MIC). The mutants were 8- to 64-fold more resistant to ciprofloxacin and showed complete cross resistance to nalidixic acid, ofloxacin, enoxacin, and norfloxacin. Two chromosomal resistance genes, cfxA and cfxB, were mapped between eda-9001 and phe-2 and near pyrB52 distal to proC130, respectively. The cfxB mutation was identical to a nalB mutation and conferred cross resistance to novobiocin, tetracycline, carbenicillin, and chloramphenicol, suggesting that there is an effect on permeability. DNA gyrase A and B subunits were purified from strain PAO2 (wild type), PAO236 nalA2, PAO4704 cfxA2, and PAO4700 cfxA1 cfxB1. Inhibition of gyrase-mediated DNA supercoiling by ciprofloxacin or nalidixic acid was greatly reduced in preparations derived from each of the mutants. Inhibition studies on reconstituted heterologous gyrase subunits showed that decreased inhibition was dependent on the mutant gyrase A subunit. We conclude that ciprofloxacin resistance in P. aeruginosa PAO2 can occur by mutation in the nalB gene or the gene for DNA gyrase A (formerly nalA).

Posttranscriptional defects in beta-globin messenger RNA metabolism in beta-thalassemia: abnormal accumulation of beta-messenger RNA precursor sequences.

The production of beta-globin messenger RNA (mRNA) in beta-thalassemic erythroblasts was studied during pulse-chase incubations with [3H]uridine. Globin [3H]mRNA was quantitated by molecular hybridization to recombinant DNA probes complementary to globin mRNA and mRNA precursor sequences. Each of six patients with beta +-thalassemia produced normal amounts of globin alpha and beta [3H]mRNA during a 20-min pulse incubation, but the beta/alpha [3H]mRNA ratio declined to steady-state levels during a chase incubation, suggesting posttranscriptional defects in beta-globin mRNA metabolism. beta-globin mRNA precursor production was estimated by measurement of [3H]RNA sequences hybridizing to a pure DNA probe containing only the large intervening sequence (intron) of the beta-mRNA precursor. Four of the patients exhibited abnormal accumulation of 3H-beta-intron sequences (2-10 times normal), indicating abnormal posttranscriptional processing. In the remaining two patients, one of whom is known to carry a mutation in the small intron of the beta-globin gene, accumulation of large 3H beta-intron RNA and beta-globin [3H]mRNA was normal in nuclei, but the ratio of beta/alpha [3H]mRNA in cytoplasm was reduced, suggesting a different posttranscriptional defect in beta-mRNA processing. These findings imply the existence of heterogeneous posttranscriptional abnormalities in beta-globin mRNA metabolism in different patients with beta-thalassemia. The initial rates of gamma- and delta-mRNA synthesis were low in all patients, suggesting that the low level of expression of these genes in adults is mediated at the transcriptional level.

[Nutritional surveys in some centers of 3 provinces of Sardinia. II. Nutrition and tradition]. / Rilevamenti alimentari in alcuni centri di tre province della Sardegna: Nota II Alimentazione e tradizione.

The differences between the nutritional habits we noticed in the villages under study, concern the geographical position, the kind of agricultural or pastoral economy and industrialization. However, these are not the only decisive factors. In some villages where average income in high, tradition still influences nutritional habits, as the preference for some typical dishes suggests. In other villages, where income is low, but where progress has been rapidly reached, the preference for more sophisticated or advertised foods seems to prevail.

[Influence of a high protein diet on the urinary and fecal excretion of calcium in albino rats]. / Influenza della dieta iperproteica sull'escrezione urinaria e fecale del calcio in ratti albini.

Albino rats (Sprague-Dawley) of mean weight 100 g were divided into four groups and given for 7 days a balanced diet. They were then placed in metabolic cages for fifteen days and fed diets containing different quantities of casein: 18% (D18), 36% (D36), 50% (D50) and 72% (D72). The levels of total calcium, inorganic phosphorus, alkaline phosphatase activity, total proteins and urea were determined. The urinary and fecal excretion of calcium were determined on specimens of urine and stool collected every two days. The metabolic balance of nitrogen was also estimated. The results show there is not a linear relationship between a high protein diet and plasma protein levels, but a progressive body calcium loss was observed with the increase of casein in the diet, which confirms what other workers have already suggested.

[Uptake of retinol-C14 into different tissues of vitamin A deficient albino rats]. / Incorporazione di retinolo-C14 in differenti tessuti di ratti albini a dieta priva di vitamina A.

Groups of rats were fed a vitamin A free diet for 30, 60 and 90 days. They were then administered orally a single dose of 1,25 microC of retinol-C14. After 3 h, 3, 6 and 12 days from the administration, the incorporation into the different tissues was determined. The results show that the incorporation decreases, on the average, as the experimental period and the intervals after the administration of retinol-C14 grow longer.

[Uptake of retinol-C14 into liver mitochondrial membranes and erythrocyte stroma of albino rats on a vitamin A-free diet]. / Incorporazione di retinolo-C14 nelle membrane dei mitocondri epatici e nello stroma dei globuli rossi di ratti albini a dieta priva di vitamina A.

We have determined the incorporation of retinol-C14 into liver mitochondrial membranes and blood cell stroma in albino male Wistar strain rats, fed with a vitamin A deficient diet for 30, 60 and 90 days. At the end of the vitamin A deficient period, rats were kept fasting for 24 hours and then administered orally retinol-C14 (1,25 microC). They were divided into groups and killed after 3 hours, 3, 6, 12 days from the administration of retinol-C14. The incorporation is very high in one-month deficient rats, it decreases as the experimental period and the intervals after the administration of retinol-C14 grow longer.

[Distribution and depletion with time of retinol-C14 in different tissues and organs of albino rats]. / Distribuzione e deplezione nel tempo di retinolol-C14 in differenti tessuti ed organi di ratti albini.

Albino male rats of Wistar strain, weighing about 350 g., were administered orally a single dose of 2,50 mu C of retinol-C14 after a fast of 24 h. At intervals of 3 h and of 3, 6, 12, 18 and 24 days rats were killed; livers, kidneys, blood, white fat tissue and brown fat tissue were removed. Samples were dried and radioactivity was measured. From the results, expressed in mu C/g of dry tissue, it is clear that vitamin A is taken up by all tissues. After 3 days it decreases in all tissues, between the 3rd and 18th day, its rate is constant and the depletion is complete within 24 days.