Function of the Evx-2 gene in the morphogenesis of vertebrate limbs.

Vertebrate gene members of the HoxD complex are essential for proper development of the appendicular skeletons. Inactivation of these genes induces severe alterations in the size and number of bony elements. Evx-2, a gene related to the Drosophila even-skipped (eve) gene, is located close to Hoxd-13 and is expressed in limbs like the neighbouring Hoxd genes. To investigate whether this tight linkage reflects a functional similarity, we produced a null allele of Evx-2. Furthermore, and because Hoxd-13 function is prevalent over that of nearby Hoxd genes, we generated two different double mutant loci wherein both Evx-2 and Hoxd-13 were inactivated in cis. The analysis of these various genetic configurations revealed the important function of Evx-2 during the development of the autopod as well as its genetic interaction with Hoxd-13. These results show that, in limbs, Evx-2 functions like a Hoxd gene. A potential evolutionary scenario is discussed, in which Evx-2 was recruited by the HoxD complex in conjunction with the emergence of digits in an ancestral tetrapod.

An apparent autocrine mechanism amplifies the dexamethasone- and retinoic acid-induced expression of mouse lipocalin-encoding gene 24p3.

We have isolated, sequenced and characterized the mouse 24p3 gene. The 24p3 protein is a member of the lipocalin family comprising secreted transporters of hydrophobic ligands. The 24p3 cDNA had been initially isolated during a search for genes overexpressed during a SV40-induced mitotic reaction [Hraba-Renevey et al., Oncogene 4 (1989) 601-608]. 24p3 comprises six exons, five introns and 793 bp of 5' regulatory region. The transcription start point (tsp) was identified by primer extension. Putative regulatory elements, including a TATA-like box and two glucocorticoid responsive core elements (GRE), have been mapped in the 5'-flanking region. Based on this observation, we examined the effect of a glucocorticoid (dexamethasone, Dex) on 24p3 expression. Dex induced the expression of 24p3 dramatically in the absence of de novo protein synthesis. This activation was further amplified by an apparent autocrine mechanism. Similar results were obtained with retinoic acid. Using the cat reporter gene system, we have shown that the 5'-flanking region of 24p3 confers Dex inducibility. Furthermore, we have identified a 43-bp region of the 24p3 promoter required for the Dex responsiveness. The biological implications are discussed in light of these results.

SV40-induced expression of mouse gene 24p3 involves a post-transcriptional mechanism.

SV40 and polyoma virus induce a mitotic host reaction in confluent, Go-arrested primary mouse kidney cell cultures. To define the primary effects of infection we constructed a cDNA library corresponding to polyA+ mRNA isolated shortly after onset of polyoma T-antigen synthesis. By differential screening of the library we have isolated and then sequenced cDNA recombinant 24p3; determined by Northern blotting, 24p3 mRNA steady state levels increased in parallel with polyoma and SV40 T-antigen synthesis. Since this rapid and early increase was particularly striking (14-20 fold) in SV40-infected cells, we studied the molecular mechanism of induction in this virus-cell system. We show that wt SV40 large T-antigen is required for the increase in 24p3 mRNA levels. The results tend to exclude that this increase is due to an SV40-induced stabilization of the 24p3 mRNA, or to an SV40-induced stimulation of transcription of the 24p3 gene; they are compatible with the working hypothesis that SV40 large T-antigen increases the efficiency of processing, possibly splicing, of the 24p3 pre-mRNA. The biological implications of these results are discussed.

Expression of a mouse replacement histone H3.3 gene with a highly conserved 3' noncoding region during SV40- and polyoma-induced Go to S-phase transition.

We have isolated and sequenced a mouse replacement variant histone H3.3 cDNA. It corresponds to the most abundant mRNA expressed from a unique gene by the use of one out of three polyadenylation sites. The 3' non coding region of H3.3 is very long (approximately 1100 nt) and highly conserved throughout evolution since it is about 95% homologous to the 3' non coding region of the chicken H3.3B gene. We studied the expression of the H3.3 gene during SV40- and polyoma-induced mitotic host reaction in confluent, Go-arrested primary mouse kidney cell cultures. H3.3 replacement variant mRNA steady state levels increased during the Go to S-phase transition, apparently as the result of two mechanisms: one related to cell growth, whereas the other was linked to cellular DNA synthesis. The latter mechanism was however far less pronounced than with replication histone variant mRNAs. The biological implications of these results are discussed.