Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics.

The complete 1,751,377-bp sequence of the genome of the thermophilic archaeon Methanobacterium thermoautotrophicum deltaH has been determined by a whole-genome shotgun sequencing approach. A total of 1,855 open reading frames (ORFs) have been identified that appear to encode polypeptides, 844 (46%) of which have been assigned putative functions based on their similarities to database sequences with assigned functions. A total of 514 (28%) of the ORF-encoded polypeptides are related to sequences with unknown functions, and 496 (27%) have little or no homology to sequences in public databases. Comparisons with Eucarya-, Bacteria-, and Archaea-specific databases reveal that 1,013 of the putative gene products (54%) are most similar to polypeptide sequences described previously for other organisms in the domain Archaea. Comparisons with the Methanococcus jannaschii genome data underline the extensive divergence that has occurred between these two methanogens; only 352 (19%) of M. thermoautotrophicum ORFs encode sequences that are >50% identical to M. jannaschii polypeptides, and there is little conservation in the relative locations of orthologous genes. When the M. thermoautotrophicum ORFs are compared to sequences from only the eucaryal and bacterial domains, 786 (42%) are more similar to bacterial sequences and 241 (13%) are more similar to eucaryal sequences. The bacterial domain-like gene products include the majority of those predicted to be involved in cofactor and small molecule biosyntheses, intermediary metabolism, transport, nitrogen fixation, regulatory functions, and interactions with the environment. Most proteins predicted to be involved in DNA metabolism, transcription, and translation are more similar to eucaryal sequences. Gene structure and organization have features that are typical of the Bacteria, including genes that encode polypeptides closely related to eucaryal proteins. There are 24 polypeptides that could form two-component sensor kinase-response regulator systems and homologs of the bacterial Hsp70-response proteins DnaK and DnaJ, which are notably absent in M. jannaschii. DNA replication initiation and chromosome packaging in M. thermoautotrophicum are predicted to have eucaryal features, based on the presence of two Cdc6 homologs and three histones; however, the presence of an ftsZ gene indicates a bacterial type of cell division initiation. The DNA polymerases include an X-family repair type and an unusual archaeal B type formed by two separate polypeptides. The DNA-dependent RNA polymerase (RNAP) subunits A', A", B', B" and H are encoded in a typical archaeal RNAP operon, although a second A' subunit-encoding gene is present at a remote location. There are two rRNA operons, and 39 tRNA genes are dispersed around the genome, although most of these occur in clusters. Three of the tRNA genes have introns, including the tRNAPro (GGG) gene, which contains a second intron at an unprecedented location. There is no selenocysteinyl-tRNA gene nor evidence for classically organized IS elements, prophages, or plasmids. The genome contains one intein and two extended repeats (3.6 and 8.6 kb) that are members of a family with 18 representatives in the M. jannaschii genome.

An insertional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin nice (beta 220/216).

Spectrin Nice (beta 220/216) is a spectrin variant associated with a shortened beta chain found in a patient with elliptocytosis. The shortened beta chain (beta' chain) appeared as an additional band of approximately 216 Kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was defective in its ability to be phosphorylated. There were increased amounts of spectrin dimers in crude spectrin extracts from the propositus and the association constant of spectrin dimer self-association was decreased. There was an associated increase of the alpha I 74-Kd fragment from the alpha chain after partial trypic digestion of spectrin. To identify the underlying molecular defect, we analyzed cDNA for beta spectrin obtained by polymerase chain reaction amplification of reverse-transcribed reticulocyte messenger RNA from peripheral blood of the propositus. DNA sequencing of individual as well as pooled subclones showed that two extra bases (GA) are inserted in codon no. 2046 in one allele of the beta-spectrin gene. The insertion results in a frameshift mutation and generates an aberrant C-terminus truncated by about 4 Kd, consistent with the estimated size of the beta' chain observed. By allele-specific oligonucleotide hybridization, the insertion was shown to be present in the propositus and absent in his parents, confirming a previous proposal that it is a de novo mutation. The determination of the location of the mutation in spectrin Nice points to specific regions of the beta-spectrin chain where phosphorylation may occur. A model is proposed to describe the interaction between the alpha- and beta-spectrin chains and to explain the effects of the mutation found in spectrin Nice on the trypsin digestion pattern of its associated alpha chain.

Sp alpha V/41: a common spectrin polymorphism at the alpha IV-alpha V domain junction. Relevance to the expression level of hereditary elliptocytosis due to alpha-spectrin variants located in trans.

Spectrin alpha-chain mutants associated with hereditary elliptocytosis are highly variable in their level of expression. It has been assumed that the degree of elliptocytosis can be increased when the spectrin alpha chain, encoded by the alpha gene in trans to the variant, is expressed at a low level. We now provide strong evidence for the existence of low-level expression of spectrin alpha chains. This condition is referred to as the alpha V/41 polymorphism. It has been observed in 15 different families or individuals of French, North African, and African ancestry in which seven distinct elliptocytogenic alpha-spectrin variants were co-inherited. Whenever the alpha V/41 polymorphism was present, the severity of the biochemical, morphological, and, sometimes, the clinical phenotype of elliptocytosis was increased. The alpha V/41 polymorphism was also frequently encountered among 36 unrelated control subjects in the heterozygous or homozygous states, and was entirely asymptomatic in both cases. The main biochemical feature was an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction. Alteration of the facing beta IV domain of spectrin was demonstrated by in vitro spectrin dimer reconstitution experiments. It appears that the alpha V/41 polymorphism is often required for alpha-spectrin elliptocytogenic variants to become manifest in the heterozygous state. Thus, alpha-spectrin-related elliptocytosis may be viewed as a bifactorial condition.

Occurrence of the alpha I 22 Arg----His (CGT----CAT) spectrin mutation in Tunisia: potential association with severe elliptopoikilocytosis.

A category of spectrin alpha I domain variants are manifested by the increase of the alpha I 74 kDa fragment at the expense of the parent 80 kDa fragment following partial tryptic digestion. We describe a particular case of alpha I/74 abnormality in a Tunisian family. The propositus was severely ill and had an elliptopoikilocytosis. To the contrary, his father, who carried the same alpha I/74 variant, displayed no clinical signs and a few elliptocytes. The increase of the alpha I 74 kDa fragment was more pronounced in the propositus than in his father. Unexpectedly, the spectrin content was reduced to similar (and limited) extents in both of them, and the father displayed nearly as pronounced an increase of the spectrin dimer percentage as the propositus following low ionic strength extraction. In vitro spectrin dimer reconstitution experiments indicated that the primary mutation was located in the alpha-chain itself (not in the beta-chain as is the case in some alpha I/74 mutants). Following polymerase chain reaction (PCR) amplification, cloning and sequencing of exon 2 of spectrin alpha-gene in the father, we found the G----A substitution at position 2 of codon 22 (CGT----CAT; Arg----His). This mutation has been recently discovered in a family of French descent. Dot blot hybridization confirmed that the substitution was transmitted with the alpha I/74 abnormality. As previously shown, the enhancement of its expression level in the propositus, with respect to the father, was accounted for by the presence of a factor carried by the alpha-spectrin allele in trans and transmitted by the mother.

Exercise and arthritis. Kinesiology and the degenerative joint.

Advanced methods of testing allows the kinesiologist to demonstrate qualities and quantities of muscular and joint performance heretofore not available. These discerning tests shed new light on problems related to arthritis, the dilemmas of disuse atrophy, and the benefits of exercise rehabilitation. Principles of exercise for the degenerative joint are offered.

Two elliptocytogenic alpha I/74 variants of the spectrin alpha I domain. Spectrin Culoz (GGT----GTT; alpha I 40 Gly----Val) and spectrin Lyon (CTT----TTT; alpha I 43 Leu---Phe).

Spectrin alpha I/74 elliptocytosis results from abnormalities involving the "head" region of spectrin dimer. Increased susceptibility to trypsin enhances cleavage of the alpha spectrin chain, yielding an increased amount of the alpha I 74-kD fragment at the expense of the alpha I 80-kD parent fragment. Recently we showed that the mutations causing the Sp alpha I/74 abnormality may lie in the alpha- or the beta-chain, and that spectrin Culoz and spectrin Lyon were two (alpha I/74) alpha-variants, respectively. We now show that the spectrin Culoz alpha I domain undergoes prominent tryptic cleavage after Lys 42, whereas cleavage prevails after Arg 39 in spectrin Lyon. Applying the polymerase chain reaction (PCR) technique to exon 2 of the spectrin alpha I domain, we have established that the mutation responsible for spectrin Culoz is alpha I 40 Gly----Val; GGT----GTT. Applying the PCR technique to the cDNA derived from reticulocyte mRNA, we have shown that the mutation responsible for spectrin Lyon is alpha I 43 Leu----Phe; CTT----TTT. Studies of normal controls and of family members using dot blot hybridization with allele-specific oligonucleotide probes confirmed these results. Variants such as spectrin Culoz and spectrin Lyon should provide insight into a region that participates in spectrin dimer self-association and whose susceptibility to proteolysis must reflect subtle conformational changes.

A haemolytic syndrome associated with the complete absence of red cell membrane protein 4.2 in two Tunisian siblings.

We report on the complete absence of protein 4.2 in two Tunisian siblings. The propositus presented with a haemolytic anaemia that evolved in an intermittent fashion until she was cured by splenectomy. Her red cells had a normal morphology, as well as normal deformability upon osmotic gradient ektacytometry. SDS-polyacrylamide gel electrophoresis failed to reveal any protein 4.2. Using anti-protein 4.2 polyclonal antibodies. Western blots were also unable to detect protein 4.2. Preparation of inside out vesicles resulted in no detectable loss of ankyrin. The propositus's sister presented with a haemolytic anaemia but had not undergone splenectomy; she showed the same biochemical features. The two cases presented of missing protein 4.2 are the first ones to be described outside the Japanese population. Considered as homozygotes for some defect that must alter the protein 4.2 gene itself, they exemplify a unique syndrome pertaining neither to elliptocytosis nor to spherocytosis, at least not closely. The parents, who are first cousins and whom we regarded as heterozygotes, were clinically and morphologically normal; they had a normal content of protein 4.2. Therefore, the 4.2 (-) haemolytic anaemia appears as entirely recessive.

Assignment of Sp alpha I/74 hereditary elliptocytosis to the alpha- or beta-chain of spectrin through in vitro dimer reconstitution.

Partial digestion of spectrin dimers in vitro has allowed the definition of domains. For example, the portions of the dimers that are involved in spectrin self-association are represented by the alpha I and the beta I domains. The alpha I domain (80 Kd) is further cleaved into a minor 78 Kd fragment and, more substantially, into a 74 Kd fragment. The intensity of the latter, which we expressed as the 74:(80 + 78 + 74) ratio, or the 74:alpha I ratio, is variable depending on the experimental conditions, eg, in fine, on the conformation of the alpha I domain. A number of cases of hereditary elliptocytosis (HE) are associated with an increase of the 74:alpha I ratio, also referred to as the Sp alpha I/74 abnormality. Several lines of evidence have suggested that the causal mutations may lie in the alpha- or the beta-chain, a point of importance before one undertakes studies at the gene level. In order to address this question, we reconstituted spectrin dimers in vitro, combining alpha- and beta-chains of various origins, and then carried out partial digestion and assayed the Sp alpha I/74 abnormality. The patterns obtained with reconstituted dimers were nearly identical to those of native dimers. We applied the assay to three spectrin variants that cause Sp alpha I/74 HE: (1) a variant that we previously designated spectrin Nice and whose beta-chain lacks a 4 Kd fragment in its C-terminal region; and two distinct variants that we found in two unrelated white families and that we provisionally designated spectrin Lyon and spectrin Culoz. The Sp alpha I/74 abnormality appeared in all kinds of dimers that harbored the beta-chain of spectrin Nice, or the alpha-chain of spectrin Lyon or spectrin Culoz, respectively. Therefore, we confirmed that spectrin Nice is a (alpha I/74) beta-variant, and established that both spectrin Lyon and spectrin Culoz are (alpha I/74) alpha-variants. The present assay may be extended to any spectrin variant displaying the Sp alpha I/74 abnormality.

Two distinct variants of erythrocyte spectrin beta IV domain.

We report two distinct variants affecting the beta IV domain of erythrocyte spectrin, designated spectrin Saint-Chamond and spectrin Tlemcen. They were discovered in a French family and an Algerian individual, respectively. They appeared clinically and morphologically asymptomatic in the heterozygous state. In two-dimensional maps of spectrin partial digests, both mutants were manifested by cathodic shifts (with no change of the molecular weights) of the peptides that cover the N-terminal region of spectrin beta IV domain. The relevance of the abnormal peptides to the beta IV domain was established by quantitative analysis and by Western blotting using anti-beta IV domain-specific antibodies. These two variants are thus far the most distal variants of spectrin to be defined on an unequivocal structural basis.

Reduction of membrane band 7 and activation of volume stimulated (K+, Cl-)-cotransport in a case of congenital stomatocytosis.

We report on a case of congenital stomatocytosis in a French boy presenting with a haemolytic anaemia requiring splenectomy at the age of 6. The red cells included 15-20% stomatocytes and displayed a marked increase of volume. Their osmotic resistance and density were reduced; however, their deformability was unaltered in isotonicity. Erythrocyte Na+ was high (27 mEq/l) and K+ low (65 mEq/l). The newly described (K+, Cl-)-cotransporter normally triggered by hypo-osmotic stress, was activated to maximal capacity. Membrane band 7 was reduced by 72%. From anamnestic data, the condition appears to have been transmitted by the father. The mother proved to be strictly normal on clinical, morphological, osmotic and biochemical bases. We suggest that the partly missing band 7 may play an important role in the genesis of stomatocytosis.

[Interaction between various alkylxanthines and the proteins of the erythrocyte skeleton]. / Recherche d'une interaction entre certaines alkylxanthines et les protéines du squelette érythrocytaire.

We searched an interaction between (i) pentoxifylline and/or propentofylline, and (ii) the red cell membrane with special emphasis on the membrane skeleton. It appeared (i) that propentofylline has no permanent binding site on the membrane, (ii) that propentofylline and/or pentoxifylline do not detectably alter spectrin conformation (no change of spectrin dimer self-association or of spectrin limited digestion in the presence of trypsin), and (iii) that these compounds have no effect on membrane protein phosphorylation, particularly the cAMP-dependent and TPA-dependent phosphorylation of protein 4.1.

Spectrin Oran (alpha II/21), a new spectrin variant concerning the alpha II domain and causing severe elliptocytosis in the homozygous state.

We report on spectrin Oran (alpha II/21), a new spectrin variant found in an Algerian family. It was characterized by the absence of the spots that classically correspond to the alpha II domain using two-dimensional analysis of spectrin limit digests. On the contrary, the abnormal domain was represented by a new set of spots in the 21-Kd and 16-Kd regions, as demonstrated by Western blots using anti-alpha II domain polyclonal antibodies. Spectrin Oran (alpha II/21) was found in the homozygous state in two children belonging to two separate branches of the family. It yields a severe elliptocytosis. Spectrin self-association was altered. The variant was much more difficult to prove in the heterozygous state, in which it results in no clinical and virtually no morphological symptom. In all four parents involved, however, electrophoretic analysis and Western blots showed the existence of the alpha II 21-Kd and 16-Kd peptides. In one parent, who combines spectrin Oran (alpha II/21) and the alpha II type-2 polymorphism, the two-dimensional spots (52, 39, 34, and 29 Kd) were quantified and appeared reduced by 30%: there was an intermediary decrease of spectrin self-association in this person. In the three other parents, spectrin Oran combined with the alpha II type-1 polymorphism. The alpha II type-1 spots (46, 35, 30, and 25 Kd) appeared in normal range, and spectrin self-association was normal. Along with previous observations, the present data emphasize the large fluctuations of the alpha-variant percentage. Provided spectrin Oran was present in a sufficient proportion, we found an associated alteration of the beta II domain (that faces the alpha II domain in the spectrin dimer): the beta II 65-Kd fragment was reduced and the beta II 52-Kd fragment was reciprocally increased.

Spectrin Tunis (alpha I/78): a new alpha I variant that causes asymptomatic hereditary elliptocytosis in the heterozygous state.

Spectrin Tunis (alpha 1/78) was found in the heterozygous state in a young white North-African man and his mother. Both of them presented with mild elliptocytosis. Using one-dimensional electrophoresis, a sharp 78 kd fragment was present with a reciprocal decrease of the alpha I 80 kd domain. Kinetic analysis unambiguously confirmed that the 78 kd fragment developed at the expense of the alpha I 80 domain. The alpha I 74 kd peptide was not flanked with a peptide lacking a 2 kd fragment. From this fact, it could be inferred that the site for additional proteolysis is located upstream from arginyl residue 39 and, more precisely, should lie 10 to 20 amino-acid residues (-2 kd) from the alpha-chain N-terminus. The percentage of spectrin dimers in 4 degrees C extracts was high (over 40%), contrasting with the absence of clinical symptoms related to elliptocytosis. This is the first mutation responsible for elliptocytosis found in Tunisia.

Aberrant pattern of red cell membrane and cytosolic proteins in a case of congenital dyserythropoietic anaemia.

We report an unusual case of congenital dyserythropoietic anaemia (CDA). The propositus is a 25-year-old gipsy female presenting with a recessively inherited haemolytic anaemia. The diagnosis of CDA was based on erythrokinetic data and the morphological appearance of the erythroid precursors. The direct assay of HEMPAS antigen was negative. In peripheral blood there were 15% dacryocytes. The red cell membrane protein pattern was dramatically altered, with four major aberrant bands. Band a (mol wt 86,000) was at the lower edge of band 3, band b (mol wt 82,000) was below band 3, band c (68,000) and band d (67,000) were below band 4.2. In addition, there was an array of aberrant minor bands below band d. Gel densitometric determinations and immunological characterization showed that these bands did not derive from any of the major components of the membrane. In fact, membrane proteins appeared normal in many respects, although periodic acid-Schiff staining revealed an apparent decrease of sialoglycoproteins. The major aberrant bands a, b and c occur in very low amounts in controls. These bands, as well as band d, also exist in normal cytosol and were strongly increased in the propositus.

Spectrin Nice (beta 220/216): a shortened beta-chain variant associated with an increase of the alpha I/74 fragment in a case of elliptocytosis.

We describe a new spectrin variant with a truncated beta-chain. It was discovered in a 17-year-old white boy presenting with intermittent jaundice and spleen enlargement. He also displayed numerous smooth elliptocytes. On sodium dodecyl sulfate-polyacrylamide gel, the truncated beta-chain (beta'-chain) appeared as an additional band of approximately 216 kilodaltons, migrating between spectrin beta-chain and ankyrin. It represented 30% of total beta-chain. The beta'-chain reacted with an antispectrin beta-chain monoclonal antibody. It failed to become phosphorylated when ghosts were incubated in the presence of [gamma-32P] adenosine triphosphate. Whole spectrin tetramerization was defective since the amount of spectrin dimer was increased in spectrin crude extract and the association constant of the spectrin dimer self-association was decreased. Spectrin whole tetramer isolated from spectrin crude extracts contained small quantities of beta'-chain. Spectrin tryptic peptides showed an increase of the 74,000-dalton fragment at the expense of the 80,000-dalton fragment. So far, the latter abnormality has been used to characterize a number of cases of hereditary elliptocytosis or pyropoikilocytosis with no other apparent change. In the present case, we consider that the abnormality is a consequence of the beta-chain alteration. The parents seemed asymptomatic. As a result, we regard this new spectrin variant as deriving from a de novo mutation.

Red cell membrane alteration involving protein 4.1 and protein 3 in a case of recessively inherited haemolytic anaemia.

We present 2 siblings with a severe congenital haemolytic anaemia. Red cells displayed a variety of abnormal shapes, including leptocytes, schizocytes and elliptocytes. Repeatedly, skeletal protein 4.1 appeared reduced by 30%. The 4.1a/4.1b ratio was normal despite the haemolytic state. No change could be detected in spectrin, nor in sialoglycoproteins. Band 3 was denser, narrower and displaced downward. The parents, who are consanguineous, were devoid of any obvious biochemical abnormality; however, their red cells were not normal. These 2 cases with reduced protein 4.1 clearly depart from 4.1 (-) hereditary elliptocytosis. The possibility of an altered binding of protein 4.1 to some other membrane component is discussed.

Sp alpha I/65 hereditary elliptocytosis in North Africa.

The Sp alpha I/65 variant of the spectrin has been recently described in black people with hereditary elliptocytosis (HE). The present study reports on a similar Sp alpha I/65 variant in nine North African persons belonging to four unrelated families. The abnormality was associated with a variable degree of elliptocytosis. In one case, red cell morphology was normal. In the nine carriers of the biochemical abnormality, the spectrin dimer self-association was defective. The association constant was reduced: 0.65 to 1.7 X 10(5) M-1 (controls: 4.6 +/- 0.5 X 10(5) mM-1 (n = 21)); in six cases, there was a higher level of spectrin dimer in the low ionic strength extract at 4 degrees C: 13.0 to 19.7% (controls: 6.4 +/- 2.1% (n = 7)). Limited tryptic digests of spectrin from the nine persons revealed a decrease of the 80,000-dalton alpha-1 domain, and the concomitant appearance of a peptide with a molecular weight of 65,000 daltons and an isoelectric point ranging from 5.0 to 5.1. There was a correlation between the proportion of the 65,000-dalton fragments, the defect of spectrin self-association, and the extent of morphological alteration. This is the first large series concerning a spectrin abnormality in non-black persons. In North Africa, cases of HE that are not due to a protein 4.1 defect have turned out so far to be associated with the Sp alpha I/65 variant.