Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product.

Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called "residual" CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.

Rapid generation of nested chromosomal deletions on mouse chromosome 2.

Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid generation of nested deletions with Cre recombinase was developed and tested. A loxP site was targeted to the Notch1 gene on chromosome 2. A targeted line was cotransfected with a second loxP site and a plasmid for transient expression of Cre. Independent random integrations of the second loxP site onto the targeted chromosome in direct repeat orientation created multiple nested deletions. By virtue of targeting in an F(1) hybrid embryonic stem cell line, F(1)(129S1xCast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extending six or seven centiMorgans. The cytology of the deletion chromosomes were determined by fluorescent in situ hybridization. Eight deletions were cytologically normal, but the two largest deletions had additional rearrangements. Three deletions, including the largest unrearranged deletion, have been transmitted through the germ line. Several endpoints also have been cloned by plasmid rescue. These experiments illustrate the means to rapidly create and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells.

Rapid characterization of growth-arrest genes in transient transfection assays.

We developed a rapid assay for identifying growth-arrest genes to facilitate studies of cell cycle regulation. A7r5 vascular smooth muscle cells were transiently transfected with two plasmids: (i) a pMSV beta Gal reporter construct expressing beta-galactosidase (beta-gal) under transcriptional control of the murine sarcoma virus long terminal repeat; and (ii) a eukaryotic expression vector driving transcription of a potential growth inhibitory c-DNA under control of the cytomegalovirus promoter/enhancer. Twenty-four hours after transfection, cellular DNA was labeled for an additional 24 h with 5-bromo-2-deoxyuridine (BrdU) to label cellular DNA. After fixation, transfected cells were identified by histochemical staining with a beta-gal substrate, 6-chloro-3-indolyl-beta-D-galactopyranoside (i.e., Red-Gal). Transfected cells (beta-gal-positive) that traversed S phase (i.e., DNA synthesis) were quantified by indirect immunocytochemical staining for BrdU. Since autoradiography was not required to score for DNA synthesis, the length of experiments was much shorter than previously described growth-arrest assays performed with transiently transfected cells. Experiments with two growth-arrest genes, p53 and the p21 cyclin-dependent kinase inhibitor, demonstrated the utility of this assay.

Molecular cloning and localization of the human GAX gene to 7p21.

The GAX homeobox gene is expressed in the cardiovascular tissues of the adult rat, including heart, lung, kidney, and blood vessels. In the vasculature it is specifically expressed in quiescent smooth muscle cells, but its expression is rapidly down-regulated when these cells are stimulated to proliferate with mitogens. Since vascular smooth muscle cell proliferation is important in the pathology of blood vessel disorders, the human GAX gene was isolated and characterized. The human GAX cDNA was obtained by an anchored-PCR approach using cDNA templates from cardiovascular tissues and amplification primers designed from sequence information of the rat GAX cDNA and the homeodomain-containing exon of the human GAX gene. The human and rat GAX gene coding sequences are 98% conserved at the amino acid level and 83% conserved at the nucleotide level. Similar to rat, the human homolog contains a CAX trinucleotide repeat N-terminal to the homeodomain that encodes for a stretch of 17 consecutive histidine or glutamine residues. The human GAX locus was mapped by fluorescence in situ hybridization to the short arm of chromosome 7 at band p21. The human cDNA sequence will be useful for analyses of GAX gene expression in cardiovascular diseases.

Cloning and sequence analysis of homeobox transcription factor cDNAs with an inosine-containing probe.

Much effort has been directed toward the isolation and characterization of homeobox cDNAs from numerous cell types because they encode transcription factors important to many cellular processes, including pattern formation in the embryo, cell growth and cell differentiation. Many novel homeobox cDNAs have been isolated by screening libraries by hybridization with degenerate oligonucleotides designed from conserved amino acid sequences in the third helix of the homeodomain. However, the degeneracy of the genetic code necessitates that these oligonucleotides be highly degenerate, often precluding their use as sequencing primers to rapidly determine clone identity. Here we describe a screening protocol for homeobox cDNAs that utilizes a short oligonucleotide probe with inosine residues incorporated at positions of maximum codon degeneracy. This probe specifically hybridizes to many classes of homeobox transcription factor cDNAs, but its primary advantage is that it also serves as an effective sequencing primer, which allows the investigator to rapidly determine whether the clones encode a protein of interest. In a screen of 500,000 plaques of a rat aorta cDNA library by this method, we identified 13 positive plaques of which 12 were found to contain homeobox cDNAs representing 5 distinct genes, and, using this probe, it was possible to obtain initial high-quality sequence information from every clone isolated that contained a homeodomain.

Molecular cloning of a diverged homeobox gene that is rapidly down-regulated during the G0/G1 transition in vascular smooth muscle cells.

Adult vascular smooth muscle cells dedifferentiate and reenter the cell cycle in response to growth factor stimulation. Here we describe the molecular cloning from vascular smooth muscle, the structure, and the chromosomal location of a diverged homeobox gene, Gax, whose expression is largely confined to the cardiovascular tissues of the adult. In quiescent adult rat vascular smooth muscle cells, Gax mRNA levels are down-regulated as much as 15-fold within 2 h when these cells are induced to proliferate with platelet-derived growth factor (PDGF) or serum growth factors. This reduction in Gax mRNA is transient, with levels beginning to rise between 8 and 24 h after mitogen stimulation and returning to near normal by 24 to 48 h. The Gax down-regulation is dose dependent and can be correlated with the mitogen's ability to stimulate DNA synthesis. PDGF-AA, a weak mitogen for rat vascular smooth muscle cells, did not affect Gax transcript levels, while PDGF-AB and -BB, potent mitogens for these cells, were nearly as effective as fetal bovine serum. The removal of serum from growing cells induced Gax expression fivefold within 24 h. These data suggest that Gax is likely to have a regulatory function in the G0-to-G1 transition of the cell cycle in vascular smooth muscle cells.

Molecular cloning of a homeobox transcription factor from adult aortic smooth muscle.

We report here the cloning of a cDNA encoding a homeobox transcription factor from vascular smooth muscle and describe its unique pattern of mRNA expression at different stages in development. The cDNA isolated is 1576 base pairs in length not including the poly(A) tail and contains an open reading frame coding for a predicted 372-amino acid homeobox protein. During early embryogenesis, expression was detected in the neural tube with a sharp expression boundary occurring at an anterior position, in the myelencephalon, in the third and fourth branchial arches, and in vessels leading from the heart. In adults, however, transcripts were only detected in aortic smooth muscle and lung but were undetectable in cardiac or skeletal muscle, visceral smooth muscle, and many other tissues including brain. In neonates, expression was detected in the outflow tracts of the heart as well as in the cardiomyocytes. The expression pattern of this gene suggests that, although it likely has multiple roles during development, in the adult, it may participate in the control of vascular smooth muscle differentiation and proliferation.