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At least 19 recordsLinked to original sources
PubMed1990

Multiple mRNAs of rat brain alpha-crystallin B chain result from alternative transcriptional initiation.

Two major classes of mRNAs for the alpha-crystallin B chain (or alpha(B)crystallin), about 0.9 and 1.2 kilobases in length, are expressed in rat brain. To examine the structures of these mRNAs, we isolated cDNA clones from rat brain and genomic DNA from rat liver. Characterization of these clones as well as Northern blot analysis indicated that the various mRNAs differed in the lengths of their 5' leader sequences. RNase protection assays revealed that the gene for alpha-crystallin B chain contains multiple start sites. The transcriptional start sites of the longer mRNAs are preceded by a putative CAAT box and that of the shorter mRNA by a putative TATA box. The shorter mRNA encodes the alpha-crystallin B chain protein, whereas the longer mRNA contained three extra small open reading frames upstream of the AUG start codon for the protein. The shorter mRNA is abundant in lens, heart, muscle, and kidney, while the longer mRNAs are constitutively expressed at low levels in a wide variety of tissues. The shorter mRNA was increased by treatment with phorbol 12-myristate 13-acetate in rat C6 glioma cells. Since there is only a single copy of the alpha-crystallin B chain gene, our results indicate that the two classes of mRNAs are generated by alternative transcriptional initiation from different promoters and their expressions are regulated differentially.

Amino Acid Sequence↗
PubMed1992

Structural organization and stability of a thermoresistant domain generated by in vivo hydrolysis of the alpha-crystallin B chain from calf lens.

A protein fragment (M(r) approximately 9000) isolated from the cortex of nonpathological calf lenses has been structurally characterized. The polypeptide structure was well organized (39% alpha-helix, 33% beta-structure, and 28% remainder) according to the far-ultraviolet circular dichroism. The fluorescence was heterogeneous for the presence of two tryptophan classes. Structure perturbation by pH and denaturant revealed cooperative structural transitions which are characteristics of a globular organization. A single-step unfolding curve induced by Gdn-HCl (midpoint = 1.38 M Gdn-HCl) was monitored by emission maximum shift as well as by far-ultraviolet circular dichroism. This transition was analyzed as a two-state process. The standard free energy of unfolding in the absence of the denaturant, delta Go (H2O), was found to be 10.80 +/- 0.25 kJ/mol at 20 degrees C and pH 7.4. The fragment also shows an unusual thermal resistance. Its structure was unperturbed up to 90 degrees C according to the fluorescence and dichroism. This last property, its peculiar amino acid composition, and the sequence of a small segment are shared, among crystallins, only with the N-terminal region of the alpha-crystallin B chain. A search for proteolysis sites along the alpha-crystallin B chain sequence revealed that it possesses specific points for proteinase attack. These sites are particularly exposed and clustered in a very flexible region in the middle of the protein sequence. They are also well represented in the C-terminal extension of the molecule while a few are buried in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library.

We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapie-infected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of alpha-crystallin; the latter two may represent a response to stress.

Amino Acid Sequence↗
PubMedDate not supplied

Alexander's disease: a case report with brain biopsy, ultrasound, CT scan and MRI findings.

Alexander's disease is reported in a 6-month-old infant. The clinical course was characterized by hydrocephalus, irritability and psychomotor retardation, with subsequent seizures and spasticity. Findings on ultrasound examination, computerized tomography, magnetic resonance imaging and brain biopsy are presented. The glial nature of the Rosenthal fibers was confirmed by immuno-gold staining. Clinical classification and differential diagnosis are discussed.

Astrocytes↗
PubMed1976

Human alpha-crystallin-III isolation and characterization of protein from normal infant lenses and old lens peripheries.

Alpha-crystallin isolated from the peripheries of old normal or cataractous lenses appears to be identical, consisting of eleven polypeptides, five B, and six A chains. In contrast, alpha-crystallin isolated from normal six-week-old human lenses has only three major polypeptides, corresponding to B1, A1, and A2 of the old human lens protein as well as small amounts of some of the other components. Comparisons with bovine alpha-crystallin are also reported. Based on gel filtration experiments with Bio-Gel A-1.5m, two distinct populations of alpha-crystallin were found in old lens periphery, one containing species greater than 1.5 X 10(6) daltons and another of approximately 9 X 10(5) daltons. In the cataract preparations, the higher molecular weight fraction is predominant. This fraction is not present in young lenses.

Aged↗
PubMed1978

The quaternary structure of bovine alpha-crystallin. Size and charge microheterogeneity: more than 1000 different hybrids?

Cortial alpha-crystallin was size-fractionated by gel filtration on Ultrogel AcA22 and charge-fractionated by anion-exchange chromatography on DE-52 DEAE-cellulose using gradient elution. Electron microscopy demonstrates that both native and reassociated alpha-crystallin are heterogeneous populations of spherical or slightly ellipsoidal molecules with diameters of 13.5--16.0 nm (maximum at 14.0--15.0 nm) for native alpha-crystallin and 8.5--12.5 nm (maximum at 10.0--10.5 nm) for reassociated alpha-crystallin. An enormous charge heterogeneity of native alpha-crystallin was detected, which is shown to arise from variations in the stoichiometry of the 5 main types of subunits. The molar ratio of acidic chains (A2, A1 and A1/2-151) to basic chains (B2 and B1) varies from 70/30--80/20 (averaging about 3/1) and the amount of deamidated chains (A1 and B1) varies from 7--37%. Recombination of the subunits, after dissociation in 6 M urea, leads to a charge heterogeneity of reassociated alpha-crystallin very similar to that of native alpha-crystallin. Therefore, specific formation of pure A or B chain aggregates is not preferred. Instead, random combination of subunits is theoretically shown to be sufficient to describe the observed charge microheterogeneity of both reassociated and native alpha-crystallin. No obvious relationship exists between size and charge heterogeneity. Within these ranges of molecular weight and subunit composition there are more than 1000 different combinations of A2, A1, A1/2-151, B2 and B1 conceivable.

Animals↗
PubMed1976

Intracellular degradation and deamidation of alpha-crystallin subunits.

The subunites AA2 and N2, present in alpha-crystallin from the nucleus of young and old bovine lenses, were isolated and characterized. It was found that AA2 is identical to a shorter A-chain and hence it was designated as A1-101. The subunit N2 turned out to be identical with a shorter B-chain and was designated as b 1/2-170. Characterization of the subunit N1, present only in alpha-crystallin from the cortex of old bovine lenses, suggested that his subunit is a modified B-chain, probably a deamidation product; it was designated as B0. Con relation to the age of the fiber cells in old bovine lenses with that in calf lenses revealed that the observed specific limited degradation of the subunits of alpha-crystallin increased with older age of the tissue. The deamidation process was found not to be related to the aging of the tissue. Eventually, a clear picture concerning the heterogeneity and fickleness of the alpha-crystallin subunit structure was obtained.

Age Factors↗
PubMed1991

Photooxidation of specific residues in alpha-crystallin polypeptides.

Singlet oxygen is a biologically important, photochemically generated species that preferentially oxidizes His, Trp, and Met residues of protein molecules. Calf alpha-crystallin was photooxidized with use of meso-tetra(p-sulfonatophenyl)porphyrin (TPPS) and uroporphyrin (UP) as singlet oxygen generators. The effects of photooxidation were monitored by analyzing the changes in alpha-crystallin peptide maps obtained by reversed-phase HPLC using a photodiode array absorbance detector. The reaction led to the loss of six specific peptides, five of which contained photooxidizable residues. Peptides containing His-97 and His-154 from the A chain and Met-68 from the B chain are preferentially photooxidized, suggesting that those residues have access to singlet oxygen. Trp residues in the N-terminal region are converted to NFK, whereas Trp-60 in the B chain is not photooxidized strongly suggesting that the former are close to the surface of alpha-crystallin while the latter Trp residue is buried. Only one peptide that is lost from the peptide maps does not contain a photooxidizable group; however, this peptide does contain an apparently undigested Lys residue. It is suggested that it forms a cross-link with a photooxidized His residue.

Amino Acid Sequence↗
PubMed1977

Isolation and characterization of initiation fragments from lens 10S and 14S alpha-crystallin messenger ribonucleic acids.

The alpha-crystallin 10S and 14S messenger ribonucleic acids (mRNAs) for the B and A chains, respectively, were isolated from calf lenses. Initiation complexes were formed with both mRNAs after which the unprotected regions were digested with ribonuclease T1. A single fragment of approximately 45 nucleotides was obtained from both the 10S and 14S mRNAs. The fragments retained the ability to reform initiation complexes under standard conditions. Two-dimensional fractionation of ribonuclease T1 digests indicated considerable similarity between the 10S and 14S fragments. However, marked differences in the (U)G region were observed. The addition of the methylating agent S-adenosyl-L-methionine to the mRNA initiation system increases complex formation form two to five times, suggesting that methylation may be required for initiation.

Animals↗
PubMed1976

Human alpha-crystallin: characterization of the protein isolated from the periphery of cataractous lenses.

alpha-Crystallin has been isolated from the peripheral region of old cataractous lenses. It was found to be closely related to bovine alpha-crystallin and to human newly synthesized alpha-crystallin in terms of its amino acid composition, the size of its polypeptide chains and the lack of free NH2-terminal groups. However, in contrast to the simple urea gel electrophoretic polypeptide patterns obtained with the reference proteins, 11 polypeptides were detected in the preparation. Ten of the polypeptides were isolated and shown to be either A or B chains on the basis of their amino acid compositions and comparison of the peptide maps of their tryptic hydrolysates. The four B chains as well as the six A chains were closely related, with most of the tryptic peptides being common to all members of their respective group. A nomenclature based upon the urea gel electrophoretic mobilites of the polypeptides has been proposed to define each chain. It was found that this alpha-crystallin preparation is composed of at least two populations of macromolecules, one of which contains macromolecules greater than 5 X 10(6) daltons on the basis of gel filtration with Bio-Gel A-5m. The compositions of the two fractions were found to be essentially identical.

Aged↗
PubMed1986

On the antigenic relationship between the alpha A and alpha B subunits of alpha-crystallin in bovine lens.

The immunochemical reactivities of the alpha A and alpha B subunits from bovine alpha-crystallin have been compared using 5 monoclonal antibodies and 2 polyvalent antisera. Each subunit bound the same maximum amount of antibody, regardless of its source, and each subunit was able to completely displace alpha-crystallin from its antibodies. One monoclonal antibody (463-12.2) and mouse anti-alpha B polyclonal antiserum bound equally well to the two subunits; with the other monoclonal antibodies and an anti alpha-crystallin antiserum, the affinities for the alpha A chains were about 10(3) fold higher than those for the alpha beta chains. These observations indicate that the alpha A and alpha B subunits of bovine alpha-crystallin share several similar, but not necessarily identical, cross-reacting antigenic determinants. The reasons for the differences between these observations and those of other investigators are discussed.

Animals↗
PubMed1990

The alternative splicing product alpha Ains-crystallin is structurally equivalent to alpha A and alpha B subunits in the rat alpha-crystallin aggregate.

In rodents and some other unrelated mammals, alternative splicing of the alpha A-crystallin gene transcript results in the synthesis of the elongated alpha Ains-crystallin chain. This polypeptide is identical to the normal alpha A-crystallin chain of 173 residues, but contains an additional sequence of 23 amino acid residues inserted between positions 63 and 64. To determine the effects of this insert peptide, the structure of the rat alpha-crystallin aggregate and its subunits alpha A-, alpha Ains- and alpha B-crystallin was studied using fluorescence spectra, partial urea dissociation, and lactoperoxidase-catalysed iodination of surface residues. The data suggest that all alpha-crystallin subunits occupy equivalent positions in the protein aggregate, and that the insert peptide merely elongates the connecting peptide between the putative amino- and carboxyl-terminal domain of the alpha A-crystallin subunit.

Amino Acid Sequence↗
PubMed1976

Human alpha-crystallin. I. The isolation and characterization of newly synthesized alpha-crystallin.

Studies of the incorporation of 14C amino acids into human lens proteins demonstrate that an alpha-crystallin fraction takes up more than six times as much radioactivity as any other lens protein. Based on analyses with a calibrated Bio-Gel A-1.5 m column, a molecular weight of 4.9 x 10(5) +/- 5 per cent was obtained for this protein while sedimentation equilibrium analyses indicated a weight average molecular weight, Mw, of 7.5 x 10(5) +/- 4 per cent at 10,000 r.p.m. Gel electrophoresis in sodium dodecyl sulfate revealed two components with molecular weights of 22,000 and 20,000, values similar to those found with calf alpha-crystallin. Alkaline urea gel electrophoresis indicated one major polypeptide with a mobility similar to the B2 chain of calf alpha-crystallin and two major bands with mobilities between those of the calf alpha-crystallin A2 and A1 chains. Amino acid analyses of this newly synthesized alpha-crystallin gave a composition which with a few exceptions is very similar to that of calf alpha-crystallin. All three major polypeptides contained 14C amino acids. However, from the present data, it cannot be determined whether the three polypeptides were independently synthesized or a rapid transformation produced one of the labeled polypeptides in the A region. There appears to be between three and four times as many presumptive A as B polypeptides.

Amino Acids↗
PubMed1992

Expression and aggregation of recombinant alpha A-crystallin and its two domains.

The 20 kDa alpha A and alpha B subunits of alpha-crystallin from mammalian eye lenses form large aggregates with an average molecular weight of 800,000. To get insight into the interactions responsible for aggregate formation, we expressed in Escherichia coli the putative N- and C-terminal domains of alpha A-crystallin, as well as the intact alpha A-crystallin chain. The proteins are expressed in a stable form and in relatively high amounts (20-60% of total protein). Recombinant alpha A-crystallin and the C-terminal domain are expressed in a water-soluble form. Recombinant alpha A-crystallin forms aggregates comparable with alpha-crystallin aggregates from calf lenses, whereas the C-terminal domain forms dimers or tetramers. The N-terminal domain is expressed in an initially water-insoluble form. After solubilization, denaturation and reaggregation the N-terminal domain exists in a high molecular weight multimeric form. These observations suggest that the interactions leading to aggregation of alpha A-crystallin subunits are mainly located in the N-terminal half of the chain.

Base Sequence↗
PubMed1992

Biophysical characterization of alpha-crystallin aggregates: validation of the micelle hypothesis.

The size of alpha-crystallin aggregates, as well as the structural organization of each particle's subunits, is currently unknown, although a number of different laboratories have suggested both structures and average molecular weights (Thomson, J.A. and Augusteyn, R.C. (1984) Proc. Int. Soc. Eye Res. 3, 152). One hypothesis, compatible with literature reports and consistent with what is known of subunit primary structure and physiological function, is that alpha-crystallin exists in vivo as a naturally occurring protein micelle (Sen, A.C. and Chakrabarti, B. (1991) Biophysical J. 59, 108a.) To test this hypothesis, experiments were performed on this protein to determine its behavior under increased hydrostatic pressure and the effect of its concentration on aqueous surface tension. With increasing hydrostatic pressure, the turbidity of an alpha-crystallin solution increases exponentially to a plateau at about 6000-8000 psi; upon release of pressure, the samples slowly return to their original turbidity level. Other naturally aggregating proteins, such as skeletal muscle myosin, demonstrate a decrease in turbidity under the same conditions. The surface tension of alpha-crystallin in aqueous solution decreases to a plateau with increasing protein subunit concentration, with an inflection point over the range 0.18-0.25 mM; cholate and other amphiphiles exhibit similar behavior. In contrast, plots of surface tension over the equivalent concentration range for other protein aggregates in the same buffer more closely approximate the types of curve obtained with short chain aliphatic acids. These results indicate that alpha-crystallin behaves like the protein version of a micelle.

Animals↗
PubMed1991

Mapping of the Pim-1 oncogene in mouse t-haplotypes and its use to define the relative map positions of the tcl loci t0(t6) and tw12 and the marker tf (tufted).

Pim-1 is an oncogene activated in mouse T-cell lymphomas induced by Moloney and AKR mink cell focus (MCF) viruses. Pim-1 was previously mapped to chromosome 17 by somatic cell hybrids, and subsequently to the region between the hemoglobin alpha-chain pseudogene 4 (Hba-4ps) and the alpha-crystalline gene (Crya-1) by Southern blot analysis of DNA obtained from panels of recombinant inbred strains. We have now mapped Pim-1 more accurately in t-haplotypes by analysis of recombinant t-chromosomes. The recombinants were derived from Tts6tf/t12 parents backcrossed to + tf/ + tf, and scored for recombination between the loci of T and tf. For simplicity all t-complex lethal genes properly named tcl-tx are shortened to tx. The Pim-1 gene was localized 0.6 cM proximal to the tw12 lethal gene, thus placing the Pim-1 gene 5.2 cM distal to the H-2 region in t-haplotypes. Once mapped, the Pim-1 gene was used as a marker for further genetic analysis of t-haplotypes. tw12 is so close to tf that even with a large number of recombinants it was not possible to determine whether it is proximal or distal to tf. Southern blot analysis of DNA from T-tf recombinants with a separation of tw12 and tf indicated that tw12 is proximal to tf. The mapping of two allelic t-lethals, t0 and t6 with respect to tw12 and tf has also been a problem.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Lens structures exist transiently in development of transgenic mice carrying an alpha-crystallin-diphtheria toxin hybrid gene.

The development of lens structures in transgenic mice (lnl mice) which carry the diphtheria toxin A chain-coding sequence under the control of the alpha-crystallin promoter is examined here in detail. The initial stages of lens development during embryonic days 10.5 to 12.5 (E10.5-E12.5), including the invagination of the surface ectoderm to form a lens vesicle, closure of the vesicle to form a lens cup, and initial appearance of the lens itself, appeared identical in histologic analyses of lnl mice and genotypically wild-type littermate controls. However, by E12.5, cells in the central posterior lens of developing lnl mice appeared to be vacuolated and undergoing necrosis. This necrosis was quite prominent at E14.5 and the overall lens size was significantly reduced. The lenses of lnl mice continued to be present but were significantly smaller throughout embryonic development. The cells of these lenses were capable of undergoing biochemical differentiation, reacting with antibodies to both alpha- and beta-/gamma-crystallin. alpha-Crystallin expression was initiated at the appropriate time (E10.5) and maintained in most cells of lnl lenses. The expression of beta-/gamma-crystallins was surprising as these crystallins are expressed later in lens development after normal expression of alpha-crystallin and after the anticipated time of expression of the diphtheria toxin transgene. Despite extensive necrosis and cell death, lens structures persisted in lnl mice and disappeared only in the early postnatal period between days 3 and 6. Throughout the perinatal period, the remaining lens cells expressed both alpha- and beta-/gamma-crystallins. Prenatal development of the retina and ciliary body was relatively normal although the eye was significantly reduced in overall size. Some additional developmental defects were noted including persistent hyaloid artery and thickened cornea. In the perinatal period the rapidly expanding retina filled the entire eye leaving essentially no anterior or posterior chamber. These results clearly indicate that lens cells which are the target of diphtheria toxin-mediated cell ablation techniques persist for a significant time during development and thus place limitations on the interpretations of results obtained using this technique.

Animals↗