Molecular analysis of elements inserted into mouse gamma-actin processed pseudogenes.

DNA from ten mouse genomic clones, each containing distinct gamma-actin processed pseudogenes, was subjected to electron microscopic heteroduplex analysis, and in three cases (lambda mA36, lambda mA118 and lambda mA119) the heteroduplex formed with the DNA of a reference clone was found to be interrupted by a single-stranded loop. The genomic regions corresponding to these loops were subjected to structural analysis and they were found to represent different elements (IEs) inserted into the pseudogenes in a manner that gave rise to short target-site direct repeats. IE 36 (500 base-pairs in length) was found to be an intercisternal A-particle solo long terminal repeat (LTR), a 46 nucleotide region of which had undergone five-fold tandem amplification and subsequent mutation. IE 119 (501 base-pairs in length) was also a solo LTR, bearing similarity to the recently-described GLN-3 class of murine retroviral-like elements. IE 118 (865 base-pairs in length) is repeated 1000-2000 times in the mouse genome. It is not related to any known class of mobile elements, but does possess some sequence motifs that suggest it may be an LTR of a hitherto unrecognized family of retroviral-like elements. It also possesses a 26 out of 27 nucleotide identity to a region of the flanking pseudogene, suggesting that it may have suffered gene conversion.     

Aberrant axonal projections in mice lacking EphA8 (Eek) tyrosine protein kinase receptors.

We have generated mice homozygous for a mutation that disrupts the gene encoding EphA8, a member of the Eph family of tyrosine protein kinase receptors, previously known as Eek. These mice develop to term, are fertile and do not display obvious anatomical or physiological defects. The mouse ephA8/eek gene is expressed primarily in a rostral to caudal gradient in the developing tectum. Axonal tracing experiments have revealed that in these mutant mice, axons from a subpopulation of tectal neurons located in the superficial layers of the superior colliculus do not reach targets located in the contralateral inferior colliculus. Moreover, ephA8/eek null animals display an aberrant ipsilateral axonal tract that projects to the ventral region of the cervical spinal cord. Retrograde labeling revealed that these abnormal projections originate from a small subpopulation of superior colliculus neurons that normally express the ephA8/eek gene. These results suggest that EphA8/Eek receptors play a role in axonal pathfinding during development of the mammalian nervous system.     

''Solo'' large terminal repeats (LTR) of an endogenous retrovirus-like gene family (VL30) in the mouse genome.

VL30 genetic elements constitute a murine multicopy gene family that is retrovirus-like, despite the lack of sequence homology with any known retrovirus. Over one hundred copies of VL30 units are dispersed throughout the mouse genome. We report here that the mouse genome also contains 'solo' VL30 long terminal repeats (LTRs). These are structures which contain the LTR detached from the rest of the VL30 sequences. The isolation of solo LTRs from a mouse embryonic gene library with the aid of sub-genomic VL30 probes is described. Direct DNA sequencing established that the solo LTR unit is grossly similar to a standard VL30 LTR and that the LTR is flanked by a 4-base pair duplication. The analogy to the occurrence of solitary LTR units of transposable elements is discussed.     

Isolation of uracil auxotrophs of the fungus Acremonium cellulolyticus and the development of a transformation system with the pyrF gene

Acremonium cellulolyticus CF-2612 is a cellulase hyper-producing mutant that originated from A. cellulolyticus Y-94. In this study, we isolated a uracil auxotroph (strain CFP3) derived from CF-2612, and cloned a wild-type pyrF gene encoding orotate phosphoribosyl transferase (OPRTase) from Y-94. OPRTase activity was not detected in strain CFP3, which had one nucleotide substitution in its pyrF gene. The wild-type pyrF gene restored the defective growth of CFP3 on uracil-free medium, and PCR and Southern analyses revealed that wild-type pyrF was integrated into the genome. These results indicate that our transformation system for A. cellulolyticus with the pyrFgene as a selection marker was successful.     

Mutation in the betaA3/A1-crystallin encoding gene Cryba1 causes a dominant cataract in the mouse

During the mouse ENU mutagenesis screen, mice were tested for the occurrence of dominant cataracts. One particular mutant was discovered as a progressive opacity (Po). Heterozygotes show opacification of a superficial layer of the fetal nucleus, which progresses and finally forms a nuclear opacity. Since the homozygotes have already developed the total cataract at eye opening, the mode of inheritance is semidominant. Linkage analysis was performed using a set of genome-wide microsatellite markers. The mutation was mapped to chromosome 11 distal of the marker D11Mit242 (9.3 +/- 4.4 cM) and proximal to D11Mit36 (2.3 +/- 2.3 cM). This position makes the betaA3/A1-crystallin encoding gene Cryba1 an excellent candidate gene. Mouse Cryba1 was amplified from lens mRNA. Sequence analysis revealed a mutation of a T to an A at the second base of exon 6, leading to an exchange of Trp by Arg. Computer analysis predicts that the fourth Greek key motif of the affected betaA3/A1-crystallin will not be formed. Moreover, the mutation leads also to an additional splicing signal, to the skipping of the first 3 bp of exon 6, and finally to the deletion of the Trp residue. Both types of mRNA are present in the homozygous mutant lenses. The mutation will be referred to as Cryba1(po1). This particular mouse mutation provides an excellent animal model for a human congenital zonular cataract with suture opacities, which is caused by a mutation in the homologous gene.     

Expression of the zinc finger gene Gli3 is affected in the morphogenetic mouse mutant extra-toes (Xt).

Genetic analysis and homology between the phenotypic alterations of the human Greig Cephalopolysyndactyly Syndrome (GCPS) and the mouse mutant extra-toes (Xt) have suggested a dominant mutation in the same gene of both species. Recently, the GLI3 gene, a member of the Krüppel-related zinc finger genes, has been proposed as a candidate gene for GCPS. We examined the expression of the mouse Gli3 gene in both Xt mutant animals and during normal mouse development. Northern and RNAase protection analysis of embryos revealed that Gli3 expression was reduced about 50% in heterozygous Xt/+ mice and completely absent in homozygous Xt/Xt mice. In addition, in situ analysis of wild-type mice documented Gli3 expression in the developing limb and brain, structures affected in Xt mutant mice. This pattern suggests an important function of the Gli3 gene during morphogenesis.