Isolation and characterization of a gene from the DiGeorge chromosomal region homologous to the mouse Tbx1 gene.

DiGeorge syndrome, velocardiofacial syndrome, conotruncal anomaly face syndrome, and isolated and familial forms of conotruncal cardiac defects have been associated with deletions of chromosomal region 22q11.2. This report describes the identification, cloning, and characterization of the human TBX1 gene, which maps to the center of the DiGeorge chromosomal region. Further, we have extended the mouse cDNA sequence to permit comparisons between human and mouse Tbx1. TBX1 is a member of a phylogenetically conserved family of genes that share a common DNA-binding domain, the T-box. T-box genes are transcription factors involved in the regulation of developmental processes. There is 98% amino acid identity between human and mouse TBX1 proteins overall, and within the T-box domain, the proteins are identical except for two amino acids. Expression of human TBX1 in adult and fetal tissues, as determined by Northern blot analysis, is similar to that found in the mouse. Additionally, using 3 'RACE, we obtained a differentially spliced message in adult skeletal muscle. Mouse Tbx1 has been previously shown to be expressed during early embryogenesis in the pharyngeal arches, pouches, and otic vesicle. Later in development, expression is seen in the vertebral column and tooth bud. Thus, human TBX1 is a candidate for some of the features seen in the 22q11 deletion syndrome.

Ecotoxicity hazard assessment of styrene.

The ecotoxicity of styrene was evaluated in acute toxicity studies of fathead minnows (Pimephales promelas), daphnids (Daphnia magna), amphipods (Hyalella azteca), and freshwater green algae (Selenastrum capricornutum), and a subacute toxicity study of earthworms (Eisenia fostida). Stable exposure levels were maintained in the studies with fathead minnows, daphnids, and amphipods using sealed, flowthrough, serial dilution systems and test vessels. The algae were evaluated in a sealed, static system. The earthworms were exposed in artificial soil which was renewed after 7 days. Styrene concentrations in water and soil were analyzed by gas chromatography with flame ionization detection following extraction into hexane. Test results are based on measured concentrations. Styrene was moderately toxic to fathead minnows, daphnids, and amphipods: fathead minnow: LC50 (96 hr), 10 mg/liter, and NOEC, 4.0 mg/liter; daphnids: EC50 (48 hr), 4.7 mg/liter, and NOEC, 1.9 mg/liter; amphipods: LC50 (96 hr), 9.5 mg/liter, and NOEC, 4.1 mg/liter. Styrene was highly toxic to green algae: EC50 (96 hr), 0.72 mg/liter, and NOEC, 0.063 mg/liter; these effects were found to be algistatic rather than algicidal. Styrene was slightly toxic to earthworms: LC50 (14 days), 120 mg/kg, and NOEC, 44 mg/kg. There was no indication of a concern for chronic toxicity based on these studies. Styrene's potential impact on aquatic and soil environments is significantly mitigated by its volatility and biodegradability.

Evolution of mouse T-box genes by tandem duplication and cluster dispersion.

The T-box genes comprise an ancient family of putative transcription factors conserved across species as divergent as Mus musculus and Caenorhabditis elegans. All T-box gene products are characterized by a novel 174-186-amino acid DNA binding domain called the T-box that was first discovered in the polypeptide products of the mouse T locus and the Drosophila melanogaster optomotor-blind gene. Earlier studies allowed the identification of five mouse T-box genes, T, Tbx1-3, and Tbr1, that all map to different chromosomal locations and are expressed in unique temporal and spatial patterns during embryogenesis. Here, we report the discovery of three new members of the mouse T-box gene family, named Tbx4, Tbx5, and Tbx6. Two of these newly discovered genes, Tbx4 and Tbx5, were found to be tightly linked to previously identified T-box genes. Combined results from phylogenetic, linkage, and physical mapping studies provide a picture for the evolution of a T-box subfamily by unequal crossing over to form a two-gene cluster that was duplicated and dispersed to two chromosomal locations. This analysis suggests that Tbx4 and Tbx5 are cognate genes that diverged apart from a common ancestral gene during early vertebrate evolution.

Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development.

A novel family of genes, characterized by the presence of a region of homology to the DNA-binding domain of the Brachyury (T) locus product, has recently been identified. The region of homology has been named the T-box, and the new mouse genes that contain the T-box domain have been named T-box 1-6 (Tbx1 through Tbx6). As the basis for further study of the function and evolution of these genes, we have examined the expression of 5 of these genes, Tbx1-Tbx5, across a wide range of embryonic stages from blastocyst through gastrulation and early organogenesis by in situ hybridization of wholemounts and tissue sections. Tbx3 is expressed earliest, in the inner cell mass of the blastocyst. Four of the genes are expressed in different components of the mesoderm or mesoderm/endoderm during gastrulation (Tbx1 and Tbx3-5). All of these genes have highly specific patterns of expression during later embryogenesis, notably in areas undergoing inductive tissue interactions. In several cases there is complementary expression of different genes in 2 interacting tissues, as in the lung epithelium (Tbx1) and lung mesenchyme (Tbx2-5), and in mammary buds (Tbx3) and mammary stroma (Tbx2). Tbx1 shows very little overlap in the sites of expression with the other 4 genes, in contrast to a striking similarity in expression between members of the 2 cognate gene sets, Tbx2/Tbx3 and Tbx4/Tbx5. This is a clear reflection of the evolutionary relationship between the 5 genes since the divergence of Tbx1 occurred long before the relatively recent divergence of Tbx2 and 3 and Tbx4 and 5 from common ancestral genes. These studies are a good indication that the T-box family of genes has important roles in inductive interactions in many stages of mammalian embryogenesis.

Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity.

Tetrapod fore-and hindlimbs have evolved from the pectoral and pelvic fins of an ancient vertebrate ancestor. In this ancestor, the pectoral fin appears to have arisen following the rostral homeotic recapitulation of an existing pelvic appendage (Tabin and Laufer (1993), Nature 361, 692-693). Thus the basic appendage outgrowth program is reiterated in both tetrapod fore- and hindlimbs and the pectoral and pelvic fins of extant teleost fishes (Sordino et al. (1995) Nature 375, 678-681). Recently a novel family of putative transcription factors, which includes the T (Brachyury) locus, has been identified and dubbed the "T-box' family. In mice, all of these genes have expression patterns indicative of involvement in embryonic induction (Chapman et al. (1996) Dev. Dyn., in press), and four (Tbx2-Tbx5) are represented as two cognate, linked gene pairs (Agulnik et al., (1996), Genetics, in press). We now report that, whereas Tbx2 and Tbx3 are expressed in similar spatiotemporal patterns in both limbs, Tbx5 and Tbx4 expression is primarily restricted to the developing fore- and hindlimb buds, respectively. These observations suggest that T-box genes have played a role in the evolution of fin and limb morphogenesis, and that Tbx5 and Tbx4 may have been divergently selected to play a role in the differential specification of fore- (pectoral) versus hind- (pelvic) limb (fin) identity.

Outcomes management and asthma education in a community hospital: ongoing monitoring of health status.

An outcomes management system was implemented to evaluate the effectiveness of an asthma education program and to test its feasibility as a quality improvement tool. The study demonstrated the value of continuous monitoring of health status and the advantages of a multidimensional approach to outcome assessment and program evaluation.

Identification, characterization, and localization to chromosome 17q21-22 of the human TBX2 homolog, member of a conserved developmental gene family.

The T-box motif is present in a family of genes whose structural features and expression patterns support their involvement in developmental gene regulation. Previously, sequence comparisons among the T-box domains of ten vertebrate and invertebrate T-box (Tbx) genes established a phylogenetic tree with three major branches. The Tbx2-related branch includes mouse Mm-Tbx2 and Mm-Tbx3, Drosophila optomotor-blind (Dm-Omb), and Caenorhabditis elegans Ce-Tbx2 and Ce-Tbx2 and Ce-Tbx7 genes. From the localization of Mm-Tbx2 to Chromosome (Chr) 11, we focused our search for the human homolog, Hs-TBX2, within a region of synteny conservation on Chr 17q. We used Dm-Omb polymerase chain reaction (PCR) primers to amplify a 137-basepair(bp)_ product from human genomic, Chr 17 monochromosome hybrid, and fetal kidney cDNA templates. The human PCR product showed 89% DNA sequence identity and 100% peptide sequence identity to the corresponding T-box segment of Mm-Tbx2. The putative Hs-TBX2 locus was isolated within a YAC contig that included three anonymous markers, D17S792, and D17S948, located at Chr 17q21-22. Hybridization-and PCR-based screening of a 15-week fetal kidney cDNA library yielded several TBX2 clones. Sequence analysis of clone lambda omicron TBX2-1 confirmed homology to Mm-TBx2-90% DNA sequence identity over 283 nt, and 96% peptide sequence identity over 94 amino acids. Similar analysis of Hs-TBX2 cosmid 154F11 confirmed the cDNA coding sequence and also identified a 1.7-kb intron located at the same relative position as in Mm-Tbx2. Phylogenetic analyses of the T-box domain sequences found in several vertebrate and invertebrate species further suggested that the putative human TBX2 and mouse Tbx2 are true homologs. Northern blot analysis identified two major TBX2 expression fetal kidney and lung; and in adult kidney, lung, ovary, prostate, spleen, and testis. Reduced expression levels were seen in heart, white blood cells, small intestine, and thymus. These results suggest that Hs-TBX2 could play important roles in both developmental and postnatal gene regulation.

An ancient family of embryonically expressed mouse genes sharing a conserved protein motif with the T locus.

The T locus encodes a product with DNA binding activity that is likely to play a role in the development of all vertebrate organisms. We have identified and characterized a novel family of mouse genes that share a protein motif, the T-box, with the prototypical T locus. The T-box domain of the T locus co-localizes with its DNA binding activity. Each T-box gene is expressed in a unique temporal and spatial pattern during embryogenesis. Phylogenetic analysis suggests that at least three T-box genes were present in the common ancestor to vertebrates and invertebrates. Thus, members of the T-box family could have played a role in the evolution of all metazoan organisms.

Comparison of Moloney murine leukemia virus mutation rate with the fidelity of its reverse transcriptase in vitro.

The role of Moloney murine leukemia virus (MoMLV) reverse transcriptase (RT) in the generation of base substitution mutations during retroviral replication was analyzed. To that effect, the in vitro fidelity of the MoMLV RT was compared to the rate of base substitution mutations occurring during the replication of an MoMLV-based retroviral vector. Using the vector in an amber reversion assay, the base substitution mutation rate at a single locus was found to be 2 x 10(-6)/base pair in one cycle of vector virus replication. Analysis of the fidelity of the purified RT using the same template sequence revealed that, of the two mispairs (A.C and T.G) that would lead to reversion of the amber codon during replication, A.C occurs at a rate of 4.0 x 10(-6), and T.G occurs at a rate of 0.7 x 10(-4). While the rate of formation of A.C is very similar to the vector mutation rate, the rate of formation of T.G is more than 30 times higher. This discrepancy in rates suggests that there are other elements in the infected cells that contribute to the fidelity of viral replication.

An SOS-inducible defective retronphage (phi R86) in Escherichia coli strain B.

In Escherichia coli, RecA protein regulates the DNA damage-inducible survival-enhancing SOS response. Mutant allele recA730, which causes constitutive SOS expression, is lethal at high temperatures in B/r, a derivative of wild-type B, but not in K-12 or in certain B/r--K-12 hybrids. We present evidence that killing is due to SOS induction of a defective retronphage, phi R86, which is integrated into the B/r chromosome at 19 min, but is absent in K-12. phi R86 contains retron EC-86 which encodes reverse transcriptase and a small multicopy DNA-RNA complex, msDNA-RNA. Induction of phi R86 in recA730 B/r strains results in inhibition of host DNA replication before cell death. A retronphage 'killer' gene, ORF336, when overexpressed from a plasmid, causes similar effects without SOS induction. phi R86 is not detectably u.v.-inducible in recA+ strains.

Ultraviolet photoproducts at the ochre suppressor mutation site in the glnU gene of Escherichia coli: relevance to "mutation frequency decline".

Ochre suppressor mutations induced by UV in the Escherichia coli glnU tRNA gene are CG to TA transitions at the first letter of the anticodon-encoding triplet, CAA. Premutational UV photoproducts at this site have long been known to exhibit an excision repair anomaly ("mutation frequency decline" or MFD), whereby postirradiation inhibition of protein synthesis enhances their excision and reduces suppressor mutation yields ten-fold. We sought to clarify the basis of this unique repair response by determining the spectrum of UV photoproducts on both strands of a 36 bp region of glnU which includes the anticodon-encoding triplet. We found that four different photolesions are produced within the 3 bp sequence corresponding to the tRNA anticodon: (i) on the transcribed strand, TC (6-4) photoproducts and TC cyclobutane dimers are formed in equal numbers at the site of the C to T transition, indicating that this site is a hotspot for the usually less frequent (6-4) photoproduct; (ii) on the nontranscribed strand, TT dimers are found opposite the second and third letters of the anticodon-encoding triplet, adjacent to the mutation site; and (iii) on the nontranscribed strand, an alkali-sensitive lesion other than a (6-4) photoproduct is formed, apparently at the G in the mutation site. We suggest that mutation frequency decline may reflect excision repair activity at closely spaced UV lesions on opposite strands, resulting in double-strand breaks and the death of potential mutants.

Evidence for RecA protein association with the cell membrane and for changes in the levels of major outer membrane proteins in SOS-induced Escherichia coli cells.

Membrane fractions from Escherichia coli cells expressing DNA damage-inducible (SOS) functions contain elevated quantities of RecA protein (L. J. Gudas and A. B. Pardee, J. Mol. Biol. 101:459-477, 1976). We used two-dimensional polyacrylamide gel electrophoresis to separate membrane proteins from several strains to determine whether this effect is an artifact due to contamination of membranes during preparation by the large amount of cytoplasmic RecA present in SOS-induced cells. We found that amplification of RecA+ protein without a DNA-damaging treatment does not result in increased RecA-membrane association, whether recA is depressed specifically by an operator-constitutive recA allele or coordinately with other SOS genes by a lexA mutation that inactivates their common repressor. In contrast, large amounts of RecA appear in membrane fractions from undamaged cells of an SOS-constitutive strain carrying recA730, which encodes a spontaneously SOS-activated RecA. We conclude that the increased association of RecA with the membrane fraction requires the presence of the activated form of RecA, and that this association may contribute significantly to the SOS response. We describe also striking effects of SOS expression on the levels of the outer membrane proteins OmpA, OmpC, and OmpF.