Molecular analysis of hereditary elliptocytosis with reduced protein 4.1 in the French Northern Alps

Abstract 4.1(-) hereditary elliptocytosis (HE) is a variety of elliptocytosis resulting from the reduction (heterozygosity) or the absence (homozygosity) of protein 4.1. It is nearly always encountered in its heterozygous form. It has been found among Caucasians and North Africans in a sporadic fashion. We report the study on nine family cases of 4.1(-) HE. They were recruited independently (to the exclusion of any other variety of HE) in a limited area around the city of Annecy (French Northern Alps). The mode of genetic transmission, as well as the clinical, morphologic, and protein phenotypes fully conformed to the classical description. Western blots ruled out the existence of any protein 4.1 species of abnormal size. No obvious DNA rearrangement was detectable in any of the nine families with three 4.1 cDNA probes covering the entire coding sequence and part of the flanking 5′ and 3′ untranslated sequences. On the basis of five polymorphic sites (Bgl II, 2; Pvu II, 3), we found five different haplotypes in normal members of the 4.1(-) families. 4.1(-) HE was associated with the most common haplotype in all the propositi. 4.1 mRNA was studied in four families. Dot-blot hybridization experiments and Northern blots failed to show any detectable change in three families. On the other hand, they showed a 2-kb deletion in the 4.1(-) messenger RNA 5′-moiety in one family. These findings emphasize the heterogeneity of 4.1(-) HE at the molecular level.

Download Full-text

Molecular analysis of hereditary elliptocytosis with reduced protein 4.1 in the French Northern Alps

Blood ◽

10.1182/blood.v78.8.2113.bloodjournal7882113 ◽

1991 ◽

Vol 78 (8) ◽

pp. 2113-2119 ◽

Cited By ~ 1

Author(s):

S Feddal ◽

G Brunet ◽

L Roda ◽

S Chabanis ◽

N Alloisio ◽

...

Keyword(s):

Messenger Rna ◽

Blot Hybridization ◽

Limited Area ◽

Dot Blot ◽

Western Blots ◽

Genetic Transmission ◽

Protein 4.1 ◽

Hereditary Elliptocytosis ◽

Heterozygous Form ◽

Northern Alps

4.1(-) hereditary elliptocytosis (HE) is a variety of elliptocytosis resulting from the reduction (heterozygosity) or the absence (homozygosity) of protein 4.1. It is nearly always encountered in its heterozygous form. It has been found among Caucasians and North Africans in a sporadic fashion. We report the study on nine family cases of 4.1(-) HE. They were recruited independently (to the exclusion of any other variety of HE) in a limited area around the city of Annecy (French Northern Alps). The mode of genetic transmission, as well as the clinical, morphologic, and protein phenotypes fully conformed to the classical description. Western blots ruled out the existence of any protein 4.1 species of abnormal size. No obvious DNA rearrangement was detectable in any of the nine families with three 4.1 cDNA probes covering the entire coding sequence and part of the flanking 5′ and 3′ untranslated sequences. On the basis of five polymorphic sites (Bgl II, 2; Pvu II, 3), we found five different haplotypes in normal members of the 4.1(-) families. 4.1(-) HE was associated with the most common haplotype in all the propositi. 4.1 mRNA was studied in four families. Dot-blot hybridization experiments and Northern blots failed to show any detectable change in three families. On the other hand, they showed a 2-kb deletion in the 4.1(-) messenger RNA 5′-moiety in one family. These findings emphasize the heterogeneity of 4.1(-) HE at the molecular level.

Download Full-text

Expression of alpha- and beta-tubulin genes during dimorphic-phase transitions of Histoplasma capsulatum

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.2042-2049.1989 ◽

1989 ◽

Vol 9 (5) ◽

pp. 2042-2049

Author(s):

G S Harris ◽

E J Keath ◽

J Medoff

Keyword(s):

Phase Transitions ◽

Histoplasma Capsulatum ◽

Blot Hybridization ◽

Dot Blot ◽

Dimorphic Fungus ◽

Tubulin Gene ◽

Beta Tubulin ◽

Two Dimensional Gel Electrophoresis ◽

Western Blots ◽

Alpha Tubulin

Recent investigations have confirmed the presence of one alpha-tubulin gene (TUB1) and one beta-tubulin gene (TUB2) in the dimorphic fungus Histoplasma capsulatum. In the present study, Northern blot (RNA blot) analyses revealed multiple alpha-tubulin transcripts and a single beta-tubulin transcript in the yeast and mycelial phases of the high-virulence 217B strain and low-virulence Downs strain. S1 nuclease protection assays demonstrated one initiation start site and two major stop sites for the TUB1 transcripts, suggesting that variations in 3' processing generate the alpha-tubulin messages of 2.5 and 2.0 kilobases. Dot blot hybridization experiments indicated that tubulin gene expression is developmentally regulated during the dimorphic phase transitions. alpha- and beta-tubulin mRNAs increased six- to eightfold during the yeast-to-mycelium conversion and decreased two- to threefold during the reverse transition. These changes in tubulin mRNA content coincided with major morphological events associated with H. capsulatum development. Western blots (immunoblots) of H. capsulatum yeast-specific proteins resolved by two-dimensional gel electrophoresis demonstrated a single alpha- and a single beta-tubulin isoform. Multiple tubulin polypeptides expressed in mycelia are probably products of posttranslational modifications.

Download Full-text

A study of the messenger RNA encoding pyruvate kinase of Neurospora crassa

Bioscience Reports ◽

10.1007/bf01115007 ◽

1986 ◽

Vol 6 (2) ◽

pp. 201-208

Author(s):

M. Devchand ◽

M. Kapoor

Keyword(s):

Neurospora Crassa ◽

Pyruvate Kinase ◽

Messenger Rna ◽

Blot Hybridization ◽

Dot Blot ◽

Kinase Gene ◽

S1 Nuclease ◽

Coding Regions

In Neurospora crassa, there is a single pyruvate kinase (PK) consisting of four identical subunits of ∼60k daltons. Northern and dot blot hybridization studies, using most of the yeast pyruvate kinase gene as a probe, suggest the presence of two distinct mRNA species for pyruvate kinase, separable on the basis of the length of their polyadenylated tails, by oligo(dT)cellulose chromatography. These messages are present in polysomes, immuno-precipitated by anti-PK antibodies, indicating probable translation in vivo. Fractions containing both messages were translated in vitro in the heterologous systems as well as in a homologous N. crassa lysate, the newly-synthesized PK being detected by immunoadsorption. Protection studies using S1-nuclease suggest no major structural differences in the 5′-untranslated and most of the coding regions of the two messages.

Download Full-text

Molecular basis of Sp alpha I/65 hereditary elliptocytosis in North Africa: insertion of a TTG triplet between codons 147 and 149 in the alpha-spectrin gene from five unrelated families

Blood ◽

10.1182/blood.v73.8.2196.2196 ◽

1989 ◽

Vol 73 (8) ◽

pp. 2196-2201 ◽

Cited By ~ 18

Author(s):

AF Roux ◽

F Morle ◽

D Guetarni ◽

P Colonna ◽

K Sahr ◽

...

Keyword(s):

North Africa ◽

Molecular Basis ◽

Blot Hybridization ◽

Molecular Defect ◽

Dot Blot ◽

Hereditary Elliptocytosis ◽

Black Patients ◽

Allele Specific ◽

Alpha Spectrin ◽

Pcr Method

Abstract Hereditary elliptocytosis in North Africa is frequently associated with the alpha I/65 spectrin variant, characterized by an abnormal alpha I 65-kD instead of the normal alpha I 80-kD peptide following limited trypsin digestion of whole spectrin. A similar variant (although it yielded a 68-kD fragment) has been shown recently, in two black patients, to result from the insertion of a leucyl residue at position 148 (Marchesi et al: J Clin Invest 80:191, 1987). In order to determine if the underlying molecular defect was the same in North Africans and blacks (who originate from both sides of the Sahara Desert), we performed analysis directly at the DNA level. Starting from the DNA of an Algerian alpha I/65 heterozygote in whom the mutation was associated with identifiable RFLPs, we cloned and sequenced the alpha-spectrin gene region, which includes the mutation. We thus identified an extra leucine codon (TTG) between codons 147 and 149, the coding sequence becoming CAG TTG TTG CTG instead of CAG TTG CTG. We then used the polymerase chain reaction (PCR) method and dot-blot hybridization of the amplified DNA with mutant and normal allele-specific oligonucleotides to screen the DNA from four other unrelated North African subjects with Sp alpha I/65 hereditary elliptocytosis. In all families we studied, these subjects were heterozygous for the TTG insertion. These results demonstrate that Sp alpha I/65 hereditary elliptocytosis has the same molecular basis in North Africans and blacks.

Download Full-text

Molecular basis of Sp alpha I/65 hereditary elliptocytosis in North Africa: insertion of a TTG triplet between codons 147 and 149 in the alpha-spectrin gene from five unrelated families

Blood ◽

10.1182/blood.v73.8.2196.bloodjournal7382196 ◽

1989 ◽

Vol 73 (8) ◽

pp. 2196-2201

Author(s):

AF Roux ◽

F Morle ◽

D Guetarni ◽

P Colonna ◽

K Sahr ◽

...

Keyword(s):

North Africa ◽

Molecular Basis ◽

Blot Hybridization ◽

Molecular Defect ◽

Dot Blot ◽

Hereditary Elliptocytosis ◽

Black Patients ◽

Allele Specific ◽

Alpha Spectrin ◽

Pcr Method

Hereditary elliptocytosis in North Africa is frequently associated with the alpha I/65 spectrin variant, characterized by an abnormal alpha I 65-kD instead of the normal alpha I 80-kD peptide following limited trypsin digestion of whole spectrin. A similar variant (although it yielded a 68-kD fragment) has been shown recently, in two black patients, to result from the insertion of a leucyl residue at position 148 (Marchesi et al: J Clin Invest 80:191, 1987). In order to determine if the underlying molecular defect was the same in North Africans and blacks (who originate from both sides of the Sahara Desert), we performed analysis directly at the DNA level. Starting from the DNA of an Algerian alpha I/65 heterozygote in whom the mutation was associated with identifiable RFLPs, we cloned and sequenced the alpha-spectrin gene region, which includes the mutation. We thus identified an extra leucine codon (TTG) between codons 147 and 149, the coding sequence becoming CAG TTG TTG CTG instead of CAG TTG CTG. We then used the polymerase chain reaction (PCR) method and dot-blot hybridization of the amplified DNA with mutant and normal allele-specific oligonucleotides to screen the DNA from four other unrelated North African subjects with Sp alpha I/65 hereditary elliptocytosis. In all families we studied, these subjects were heterozygous for the TTG insertion. These results demonstrate that Sp alpha I/65 hereditary elliptocytosis has the same molecular basis in North Africans and blacks.

Download Full-text

Diffusion of a particular 4.1(−) hereditary elliptocytosis allele in the French northern Alps

Journal of Biosocial Science ◽

10.1017/s0021932000020526 ◽

1993 ◽

Vol 25 (2) ◽

pp. 239-247 ◽

Cited By ~ 4

Author(s):

G. Brunet ◽

M. T. Ducluzeau ◽

L. Roda ◽

P. Lefrancois ◽

F. Baklouti ◽

...

Keyword(s):

Molecular Genetics ◽

Founder Effect ◽

Red Cell ◽

Protein 4.1 ◽

Hereditary Elliptocytosis ◽

Cell Skeleton ◽

Very High ◽

Northern Alps ◽

Common Ancestors

SummaryHeterozygous 4.1(−) hereditary elliptocytosis results from the absence of one haploid set of protein 4.1, a major component of the red cell skeleton. Two successive epidemiological investigations revealed fifteen probands in the French Northern Alps. The frequency of this disease seems to be very high in four small villages isolated in the Aravis mountains. The genealogical study shows that eleven probands share common ancestors who lived eight or ten generations ago in these villages. Thus there was probably a founder effect from one pair of ancestors, strengthened by endogamy. In contrast, four probands originate from another area and are not genealogically related. Recent results in molecular genetics support the present data.

Download Full-text

Expression of alpha- and beta-tubulin genes during dimorphic-phase transitions of Histoplasma capsulatum.

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.2042 ◽

1989 ◽

Vol 9 (5) ◽

pp. 2042-2049 ◽

Cited By ~ 18

Author(s):

G S Harris ◽

E J Keath ◽

J Medoff

Keyword(s):

Phase Transitions ◽

Histoplasma Capsulatum ◽

Blot Hybridization ◽

Dot Blot ◽

Dimorphic Fungus ◽

Tubulin Gene ◽

Beta Tubulin ◽

Two Dimensional Gel Electrophoresis ◽

Western Blots ◽

Alpha Tubulin

Download Full-text

Fetal expression of GnRH and GnRH receptor genes in rat testis and ovary

Journal of Endocrinology ◽

10.1677/joe.0.1590179 ◽

1998 ◽

Vol 159 (1) ◽

pp. 179-189 ◽

Cited By ~ 45

Author(s):

MC Botte ◽

AM Chamagne ◽

MC Carre ◽

R Counis ◽

ML Kottler

Keyword(s):

Fetal Development ◽

Messenger Rna ◽

Hormone Receptors ◽

Blot Hybridization ◽

Internal Standard ◽

Mrna Levels ◽

Dependent Manner ◽

Dot Blot ◽

Adult Rats ◽

Total Rna

The identification of gonadal gonadotropin-releasing hormone receptors (GnRH-R) and evidence of direct inhibitory effects of GnRH agonists upon steroidogenesis in adult rat gonads, lend credence to a putative intragonadal role of a locally secreted GnRH or GnRH-like peptide. Using reverse transcription-polymerase chain reaction followed by Southern blot hybridization and sequencing, we identified, both in the ovary and in the testis of fetal and adult rats, a fully processed GnRH messenger RNA (mRNA), the sequence of which, in adult testis, was identical to that found in the hypothalamus. We also detected in the testis, but not in the ovary, a transcript containing the first intron. The ontogeny of GnRH and GnRH-R gene expression was studied in rat gonads from 14.5 to 21.5 days post-coitum (dpc), using dot blot hybridization of total RNA. During this period, the levels of cyclophilin mRNA normalized to total RNA remained unchanged. Thus, we used cyclophilin as an internal standard. GnRH mRNA was detected in the ovary at 18.5 dpc, four days later than in the testis, and similar levels were found in both sexes at birth. GnRH-R mRNA was present at 14.5 dpc in the testis and at 15.5 dpc in the ovary, with the levels at 21.5 dpc being 2.4 times higher in the testis than in the ovary. GnRH and GnRH-R mRNA levels increased in both sexes in late fetal development, but this increase appeared two days sooner in the ovary compared with the testis, thus supporting the hypothesis that expression of the GnRH and GnRH-R genes is regulated in a sex-dependent manner during fetal development. In all cases, expression of GnRH and GnRH-R preceded gonadotropin receptors in the gonads and initiation of gonadotropin secretion by the pituitary.

Download Full-text

Detection of Hepatitis a Virus and other Enteroviruses in Wastewater and Surface Water Samples by Gene Probe Assay

Water Science & Technology ◽

10.2166/wst.1991.0071 ◽

1991 ◽

Vol 24 (2) ◽

pp. 267-272 ◽

Cited By ~ 12

Author(s):

S. Dubrou ◽

H. Kopecka ◽

J. M. Lopez Pila ◽

J. Maréchal ◽

J. Prévot

Keyword(s):

Surface Water ◽

Cell Cultures ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Limit Of Detection ◽

Blot Hybridization ◽

Noncoding Region ◽

Gene Probe ◽

Dot Blot ◽

Lower Efficiency

Enteroviruses were specifically detected by dot blot hybridization when using poliovirus type 1 (PV1) derived subgenomic radiolabeled cRNA probes (riboprobes) in environmental water specimens and in the cell cultures in which the viruses were amplificated. The riboprobe corresponding to the 5' noncoding sequence detected the majority of enteroviruses. Hepatitis A virus (HAV) was specifically detected by an HAV cRNA probe corresponding to the 5' noncoding region of its genome. By this test, the limit of detection of coxsackievirus B5 and echovirus 7 seeded in mineral water was 103 to 104 PFU/spot. In cell cultures, positive signals were observed in the lysates of cells infected by one PFU. Higher positive signals were obtained with a short PV1 probe (nt 221-670) corresponding to the 5' noncoding region, which is a well preserved sequence among the enteroviruses, than with PV1 genomic probe. Hybridization allowed a good detection of enteroviral RNAs in wastewater specimens, but with a lower efficiency in surface water. In this case, amplification of viruses in the cell cultures gave significant hybridization results.

Download Full-text