Rob J.M. Moormann

Concerted and divergent evolution within the rat γ-crystallin gene family☆

Abstract The nucleotide sequences of six rat γ-crystallin genes have been determined. All genes have the same mosaic structure: the first exons contain a relatively short (25 to 44 base-pair) 5′ non-coding region and the first nine base-pairs of the coding sequence, the second exons encode protein motifs I and II, while protein motifs III and IV are encoded by the third exons. The third exons also contain a 60 to 67-base-pair long 3′ non-coding region. In the γ1–2 gene, the splice acceptor site of the third exon has been shifted three base-pairs upstream. Hence, the protein product of this gene is one amino acid residue longer. The first introns, though varying in length from 85 to 100 base-pairs, are conserved in sequence. The second introns vary considerably in length (0.9 × 10 3 to 1.9 × 10 3 base-pairs) and sequence. The second exons of the genes show concerted evolution and have undergone multiple gene conversions. In contrast, the third exons show divergent evolution. From the sequences of the third exons, an evolutionary tree of the gene family was constructed. This tree suggests that three of the present genes derive directly from the genes that originated from a tandem duplication of a two-gene cluster. Two duplications of the last gene of the four-gene cluster then yielded the other three genes. Region a′ of the third exon, encoding protein motif III, is variable, while the region encoding protein motif IV (b′) is constant. We postulate that this variability in region a′ is due to a period of radiation after each gene duplication. A comparison of the rat sequences with those of orthologous sequences from other species shows that the variation in region a′ is now preserved. Hence, it might specify the specific functional property of each γ-crystallin protein within the lens.

Structural variation in mammalian γ-crystallins based on computer graphics analyses of human, rat and calf sequences 1. Core packing and surface properties

A comparison of mammalian gamma-crystallins has been made by computer-graphics model building of several gamma-crystallin sequences based on the atomic co-ordinates of the X-ray determined structure of calf gamma-II crystallin. The complete family of rat gamma-crystallins is compared together with the orthologous protein, gamma 1-2 crystallin, from rat, human and calf lens, and the orthologous protein, gamma 2-1 crystallin, from rat and human lens. In human gamma-crystallins, a major structural difference, the replacement of an arginine by a cysteine, occurs in one of the four-fold repeated folded hairpins, which may affect stability. Sequence variations involving buried residues were observed, leading to small differences in core packing of the different sequences which may be related to their regional location in the lens. Model-building studies also indicate that the surfaces of the different gamma-crystallins vary in number of exposed hydrophobic residues and ion pairs. These differences would affect protein-water interactions and therefore contribute to refractive index. A major variable region of the gamma-crystallin structures involves polar residues surrounding the inter-domain contact and the length of the polypeptide connecting the two domains. An attempt is made to correlate bovine gamma-crystallins which are known to be responsible for cold cataract with the corresponding sequences from rat lens.

Characterization of the rat γ-crystallin gene family and its expression in the eye lens☆

Abstract Rat genomic clones, which together contain all of the rat genomic γ-crystallin sequences, have been characterized. Five γ-crystallin genes are located on a contiguous DNA region, 63 × 10 3 base-pairs long. These genes, named (5′) γ1-1, γ1-2, γ2-1, γ2-2 and γ3-1 (3′), are all oriented head to tail. A sixth γ-crystallin gene, named the γ4-1 gene, could not be linked to the γ-crystallin gene cluster with our present set of genomic clones. Mapping experiments using single copy sequences which form the extreme 5′ or 3′ region of the gene cluster showed that, if the γ4-1 gene is located on the same chromosome, then it must be separated from the gene cluster by at least 25 × 10 3 base-pairs of DNA. All γ-crystallin genes have a similar mosaic structure. They contain a large (0.9 × 10 3 to 1.88 × 10 3 base-pairs) intron in the middle of the gene and are further interrupted close to the 5′ end of the gene. The length of the first exon varies from about 40 to about 50 basepairs. The complementary DNA clone pRL-γ-3 used in this study is a copy of the transcript of the γ3-1 gene, while the second complementary DNA clone, pRL-γ-2, is most likely a copy of the transcript of the γ2-1 gene. It is further shown that rat lens messenger RNA protects fragments from the 3′ ends of the four other γ-crystallin genes against degradation by S 1 nuclease, hence all six γ-crystallin genes present in the rat genome must be transcribed in the lens. Repetitive sequences were found to be present between and around the γ-crystallin genes. Mapping with cloned repetitive sequences showed that three different repeats, designated A, B and C, occur more than once in the γ-crystallin gene cluster. Repeat C is also found in the γ4-1 region. A repetitive region 3′ to the γ3-1 gene contains members of all three repeat families.

Rat lens β-crystallins are internally duplicated and homologous to γ-crystallins

Abstract The nucleotide sequence of two cloned rat lens β-crystallin cDNAs pRLβB3-2 and pRLβB1-3 has been determined. pRLβB3-2 contains the complete coding information for a β-crystallin, designated βB3, of 210 amino acid residues. pRLβB1-3 is incomplete at its 5′ end; the 5′ codogenic information which is not present in this cDNA clone was deduced from the cloned gene. pRLβB1-3 codes for a β-crystallin polypeptide, designated βB1, whose full length is 247 amino acid residues. Considerable sequence homology is noted between the amino- and carboxy-terminal halves of each protein. The two rat β-crystallins show a substantial sequence homology with each other (60%) as well as with the published sequences of rat γ-crystallin (37%) and bovine and murine β-crystallins (55 and 45%). All these proteins have a two-domain structure which, like the bovine γII-crystallin, might be folded into four remarkably similar protein motifs. Our data further indicate that the β-crystallins can be subdivided into two groups which are evolutionarily related. Both groups are, although more distantly, also related to the γ-crystallins.

The unusual structure of heat shock locus 2-48B in Drosophila hydei

SummaryWe have previously isolated a 500 bp-long cDNA clone, NO9-15, which is derived from a nuclear transcript originating from the heat shock locus 2-48B of Drosophila hydei (Peters et al. 1982). Sequence analysis shows that this clone carries 4 complete copies and 1 partial copy of a 115 bp repeat unit. The repeats are closely homologous with a maximal sequence divergence of about 10%. The sequence does not contain an open reading frame.The genomic organization of heat shock locus 2-48B, as probed with the cloned cDNA sequence NO9-15, is highly polymorphic. Four different allelic arrangements have been found in different inbred strains. A number of genomic clones isolated from region 2-48B, both in phage lambda and in cosmid vectors, all differ in length, mainly due to varying numbers of the NO9-15 repeat unit. These differences are found primarily in the proximal region of the locus.The transcribed region of these clones includes the distal sequence flanking the NO9-15 repeat as well as the NO9-15 repeat itself. An oligo A stretch was found between the distal flanking sequence and the NO9-15 repeat region.