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The nucleotide sequence of two cloned rat lens beta-crystallin cDNAs pRL beta B3-2 and pRL beta B1-3 has been determined. pRL beta B3-2 contains the complete coding information for a beta-crystallin, designated beta B3, of 210 amino acid residues. pRL beta B1-3 is incomplete at its 5' end; the 5' codogenic information which is not present in this cDNA clone was deduced from the cloned gene. pRL beta B1-3 codes for a beta-crystallin polypeptide, designated beta B1, 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 beta-crystallins show a substantial sequence homology with each other (60%) as well as with the published sequences of rat gamma-crystallin (37%) and bovine and murine beta-crystallins (55 and 45%). All these proteins have a two-domain structure which, like the bovine gamma II-crystallin, might be folded into four remarkably similar protein motifs. Our data further indicate that the beta-crystallins can be subdivided into two groups which are evolutionarily related. Both groups are, although more distantly, also related to the gamma-crystallins.

By cloning the endpoints of a DMD-associated deletion, we have "jumped" 1100 kb from pERT87-1 (DSX164) to a new locus designated J66 (DXS268), mapping distally within the Duchenne muscular dystrophy (DMD) gene. Both J66 and JBir are mapped by field-inversion gel electrophoresis and detect abnormal SfiI fragments in DMD patients and distal DMD-associated X; autosome translocations. Our long-range map extends the physical map of the DMD gene from 800 to 2000 kb (2 Mb) and increases the mapped portion of Xp21 to approximately 8 Mb. The position of the glycerol kinase gene and the adrenal hypoplasia locus are further confined to the region between J66 and the nearest distal probe L1-4. This region spans at least 1.5 Mb. The multiallelic J66 polymorphism has immediate application in the diagnosis of DMD and generally appears to be distal to DMD mutations.

The locus DXS269 (P20) defines a deletion hotspot in the distal part of the Duchenne Muscular Dystrophy gene. We have cloned over 90 kilobase-pairs of genomic DNA from this region in overlapping cosmids. The use of whole cosmids as probes in a competitive DNA hybridization analysis proves a fast and convenient method for identifying rearrangements in this region. A rapid survey of P20-deletion patients is carried out to elucidate the nature of the propensity to deletions in this region. Using this technique, deletion breakpoints are pinpointed to individual restriction fragments in patient DNAs without the need for tedious isolation of single copy sequences. Simultaneously, the deletion data yield a consistent restriction map of the region and permit detection of several RFLPs. A 176 bp exon was identified within the cloned DNA, located 3' of an intron exceeding 150 Kb in length. Its deletion causes a frameshift in the dystrophin reading frame and produces the DMD phenotype. This exon is one of the most frequently deleted exons in BMD/DMD patients and its sequence is applied in a pilot study for diagnostic deletion screening using Polymerase Chain Reaction amplification.

The rat beta B1-crystallin gene is 13.6 kilobases long and contains six exons. The coding region of the gene is divided over five exons. Each functional entity of the protein is encoded by a separate exon except for the carboxyl-terminal extension, which shares the last exon with the fourth protein motif. Exon 2, encoding the amino-terminal extension of the protein, contains two direct repeats with an overall homology of 68% to the rat brain identifier sequence. A copy of the brain identifier sequence is also found in the 3'-flanking region of the gene. The start site of the mRNA was located by S1 nuclease mapping and analysis of the RNA sequence. The 5' end of the gene was shown to be a 27-base-pair noncoding exon, which is separated from the translation start site by 1.36 kilobases of intronic DNA. The 5'-flanking sequence of the beta B1 gene is highly homologous to that of a gamma-crystallin gene.

The sequences of a 51-kb region containing the cluster of five rat γ-crystallin-coding genes (CRYG) and of a 7-kb region surrounding the sixth rat CRYG gene were determined. Approximately 78 % of the total sequence represents intergenic DNA. We also sequenced 22 kb of DNA from the human CRYG gene cluster. All CRYG genes are associated with CpG-rich regions. The sequence similarity between the human and rat gene regions drops sharply (to 65 % ) in intronic and 3 ' -flanking regions but decreases only gradually in the 5 ' -flanking region. Highly conserved regions (>80%) are found as far upstream as 1.5kb. Overall intergenic distances are conserved. The human region contains much more repetitive DNA (24% vs. 10%) but less simple-sequence (sps) DNA (0.7% vs. 4%) than the rat region. Almost all repeats and spsDNA elements are located in the intergenic region. The location of repetitive and spsDNA differs between the orthologous regions and these elements were probably inserted after the evolutionary separation of rat and man. The Alu repeats in man and the B3 repeats in the rat are close copies of their respective consensus sequences and bordered by virtually perfect repeats. In contrast, the B1 and B2 repeats in the rat have diverged considerably from the consensus sequence and the surrounding direct repeats are usually imperfect. Thus the dispersion of the B1 and B2 repeats in the rat probably preceded that of the B3 repeats. Within the rat genomic region the spacing of Z-DNA elements is surprisingly regular, they are located about 12kb apart. A search for putative matrix-associated regions suggests that the rat CRYG gene cluster is organized into two chromosomal domains.

Application of molecular genetic techniques has greatly increased diagnostic possibilities of hereditary disorders. In 1983 the first linkage of Duchenne muscular dystrophy with flanking DNA probes was described, which made carrier detection possible in a limited number of cases. The first published prenatal diagnosis for Duchenne muscular dystrophy dates from 1985. DNA-analysis for Duchenne muscular dystrophy and Becker muscular dystrophy has become increasingly informative, firstly by the development of more flanking markers, followed by intragenic probes detecting deletions and, more recently, by the use of cDNA probes detecting a deletion or duplication mutation in over 60% of the Duchenne and Becker muscular dystrophy patients. Although these developments allow a highly reliable (greater than 99%) carrier detection and prenatal diagnosis in over 90% of cases, the continuing introduction of new probes and/or technologies has necessitated constant reappraisal of many families to derive maximum information. During the past 3 years we applied prenatal diagnosis for Duchenne and Becker muscular dystrophies with DNA-analysis on 53 male fetuses in 47 families. Twenty-two healthy male babies were born, after being diagnosed to have a low Duchenne muscular dystrophy risk. Two pregnancies also diagnosed as low risk have not yet come to term. In the other cases a high risk for Duchenne muscular dystrophy was found and the parents chose abortion. Our studies also revealed a number of important diagnostic pitfalls, such as non-paternity, karyotypic anomalies and gonadal mosaicism.

Genomic and cDNA probes were used to construct a physical map of the DMD region including the 2.3 Mb DMD gene. FIGE-analysis allows rapid screening of the complete region using only a few probes, detecting deletions or duplications in over 60% of the patients. The technique is especially powerful in the analysis of carrier females. We have found two mutational hotspots; a minor hotspot located proximally and a major hotspot within a large, centrally located intron. Deletions involving this latter intron were studied using 100 kb of cloned DNA sequences. Although breakpoints are spread over the entire region, 5 are clustered within 3 kb. Analysis of over 250 BMB/DMD families has underscored the importance of germinal mosaicism as a major diagnostic pitfall. At least 14% of new mutation cases involve germinal mosaicism and this still is a lower estimate, due to small family sizes. Hence, relatives of apparent new mutation patients should be considered to have high risk, and require appropriate counselling.