Rat lens γ-crystallins

Within the human y-crystallin gene cluster polymorphic Taq I sites are present. These give rise to three sets of allelic fragments from the -crystallin genes. Together these restriction fragment length polymorphisms define eight possi. ble haplotypes, three of which (Q, R, and S) were found in the Dutch and English population. A fourth haplotype (P) was detected within a family in which a hereditary Coppock-like cataract of the embryonic lens nucleus occurs in heterozygotes, Haplotype P was found only in family members who suffered from cataract, and all family members who suffered from cataract had haplotype P. The absolute correlation between the presence of haplotype P and cataract within this family shows that the y-crystallin gene cluster and the locus for the Coppocklike cataract are closely linked [logarithm of odds (lod) score of 7.58 at its maximum at 0 = 0]. This linkage provides genetic evidence that the primary cause of a cataract in humans could possibly be a lesion in a crystallin gene.Opacity of the lens, cataract, is the most common lens abnormality in humans. Often it is but one of the symptoms of a complex disease syndrome, and its primary cause must then be sought in the environment of the lens and not in the lens itself (1). When its occurrence is not associated with any other physical anomalies, as in some forms of hereditary cataract (for review, see ref.2), it could be due to an inherent lens defect such as a dysfunction of the genes coding for lens-specific proteins. For example, the abundant watersoluble structural lens proteins, the a-, P-, and y-crystallins, are thought to be involved in establishing the transparency of the lens and a mutation in one of these proteins might be expected to cause cataract. Unfortunately, the changes in physical properties of the lens proteins that occur during cataractogenesis are too complex to allow one to discern mutant proteins (3), and studies of cataractous lenses have therefore failed to provide direct evidence for the involvement of aberrant crystallins (or other lens-specific proteins). A direct contribution of crystallins to cataractogenesis has only been shown for coldcataract, which is due to a phase transition in the protein-water mixture in the lens (4).A difference in the population of crystallin transcripts has been noted in two murine hereditary cataracts: the lens ofthe Philly mouse lacks a P-crystallin mRNA (5), while a premature cessation of y-crystallin mRNA synthesis was seen in the lens of the CatFr mouse (6). The interpretation ofthese results in terms of the molecular lesion in these cataracts is not straightforward, however, since both cataracts are thought to be osmotic ones (2, 6). The results of the studies of these (and other) MATERIALS AND METHODSIsolation, Restriction, and Hybridization of Genomic DNA. DNA was isolated from whole blood essentially as described by Bell et al. (9). Aliquots (10 gg) were digested with Taq I under the conditions recommended by the suppliers of the enzyme (Boehringer Mannheim), and ...

supporting

confidence: 58%

A locus for a human hereditary cataract is closely linked to the gamma-crystallin gene family.

Lubsen¹,

Renwick²,

Tsui³

et al. 1987

“…In rat lens, we have found (41,42) that the yA fraction consists of three different gene products (y1-1, y4-2, y2-1), while the -yB fraction consists of the other three gene products (y2-2, 'y3-1, yy4-1). There are four potentially expressible human -y-crystallin genes (i.e., four that are not pseudogenes), so there is no a priori reason to assume that either the -yA or the -yB fraction should contain only a single polypeptide, as has been suggested in the past (45,50,54).…”

Section: Resultsmentioning

confidence: 99%

“…3. The experimental conditions used were similar to those developed by us for the charge fractionation of the multiple bovine and rat y-crystallins (41,46). Optimal tWe previously designated the y-crystallin fractions 'yH and yL (41, 42), but we now prefer to introduce the nomenclature yA and yB to avoid confusion with previous literature (14,49,(51)(52)(53) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The relative amounts of individual rat, mouse, and bovine y-crystallins, and their mRNAs, appear to vary with age, indicating that their synthesis is differentially regulated during development (9,12,(38)(39)(40)(41). In addition, there is a general shift from synthesis of y-crystallins to ,35-crystallin (9,42,43), a related monomeric protein of the 8/'y-crystallin superfamily (43,44).…”

mentioning

confidence: 92%

See 1 more Smart Citation

Human lens gamma-crystallins: isolation, identification, and characterization of the expressed gene products.

Siezen¹,

Thomson²,

Kaplan³

et al. 1987

We have isolated the individual y-crystallins expressed in young human lenses and identified with which of the six known human y-crystallin genes they each correspond.We find that at least 90% of the y-crystallins synthesized in the young human lens are the products of genes yG3 and yG4. We demonstrate that yG4-crystallin undergoes a temperaturedependent phase separation, and we have measured the lowconcentration branch of its coexistence curve (phase separation temperature vs. concentration) up to about 40 mg/ml. By comparison, we found no evidence of yG3-crystallin phase separating, even at lower temperatures and higher concentrations. This is consistent with predictions based on sequence homology between human and rat y-crystailins. The implications of these findings for human inherited and senile cataracts are considered.

“…Concentrated aqueous solutions of y-crystallins (5)(6)(7)(8) exhibit the phenomena of binaryliquid-phase separation (9)(10)(11), also known as coacervation (12). These solutions separate into two coexisting liquid phases of unequal protein concentration at temperatures less than the critical temperature for phase separation Tc.…”

mentioning

confidence: 99%

Binary-liquid phase separation of lens protein solutions.

Broide

Berland

Pande

et al. 1991

291

280

We have determined the coexistence curves (plots of phase-separation temperature T versus protein concentration C) for aqueous solutions of purified calf lens proteins. The proteins studied, calf y'Ila-, yIlb-, and yIVacrystallin, have very similar amino acid sequences and threedimensional structures. Both ascending and descending limbs of the coexistence curves were measured. We find that the coexistence curves for each of these proteins and for yIcrystallin can be fit, near the critical point, to the function W(Cc -C)/CJI = A[(Tc -T)/TCJ, where fi = 0.325, Cc is the critical protein concentration in mg/ml, T, is the critical temperature for phase separation in K, and A is a parameter that characterizes the width of the coexistence curve. We find that A andCc are approximately the same for all four coexistence curves (A = 2.6 +-0.1, Cc = 289 ± 20 mg/ml), but that Tc is not the same. For yIH-and yIIIb-crystallin, Tc7-5°C, whereas for yIIa-and yIVa-crystallin, Tc 38C. By comparing the published protein sequences for calf, rat, and human y-crystallins, we postulate that a few key amino acid residues account for the division of y-crystallins into low-Tc and high-Tc groups.The y-crystallins constitute a family of highly homologous mammalian lens proteins (1-4). Concentrated aqueous solutions of y-crystallins (5-8) exhibit the phenomena of binaryliquid-phase separation (9-11), also known as coacervation (12). These solutions separate into two coexisting liquid phases of unequal protein concentration at temperatures less than the critical temperature for phase separation Tc. From previous studies (5,7,8), it is known that location of the coexistence curve depends sensitively on the amino acid sequence of the crystallin molecule. Two distinct groups of y-crystallins have been identified in rat (7) and human (8) lenses: high-Tc crystallins and low-Tc crystallins. In these rat and human studies, the precise values of Tc for each crystallin, though inferred from the data, were not determined explicitly. For the high-Tc crystallins, only the ascending limb of the coexistence curves was measured. For the low-Tc crystallins, only an upper bound for the Tc values was established.In this paper, we report on measurements of coexistence curves for three purified calf y-crystallins-yIIIa, yIIIb, and yIVa-[in Table 2 we indicate the current nomenclature for mammalian y-crystallins (2)] and for the native calf y-crystallin mixture yIV. We have determined both the ascending and descending limbs of each coexistence curve. This information enables us to characterize the coexistence curves in detail and to determine explicitly the values of the critical concentration Cc and the critical temperature Tc for each protein. Such detailed analysis of y-crystallin phase separation, which requires gram quantities of purified protein, has been performed only for calf yII (6).We find that the purified calf y-crystallins, in accord with the purified rat and human y-crystallins, fall into two distinct groups: high-Tc (Tc > 350C) proteins, ...