MSX1 and orofacial clefting with and without tooth agenesis.

MSX1 has been considered a strong candidate for orofacial clefting, based on mouse expression studies and knockout models, as well as association and linkage studies in humans. MSX1 mutations are also causal for hereditary tooth agenesis. We tested the hypothesis that individuals with orofacial clefting with or without tooth agenesis have MSX1 coding mutations by screening 33 individuals with cleft lip with or without cleft palate (CL/P) and 19 individuals with both orofacial clefting and tooth agenesis. Although no MSX1 coding mutations were identified, the known 101C > G variant occurred more often in subjects with both CL/P and tooth agenesis (p = 0.0008), while the *6C-T variant was found more often in CL/P subjects (p = 0.001). Coding mutations in MSX1 are not the cause of orofacial clefting with or without tooth agenesis in this study population. However, the significant association of MSX1 with both phenotypes implies that MSX1 regulatory elements may be mutated.

Mutations in PITX2 may contribute to cases of omphalocele and VATER-like syndromes.

Omphalocele is a congenital anomaly with substantial morbidity. Rieger syndrome, an autosomal dominant disorder, is characterized by craniofacial abnormalities and abdominal wall defects. PITX2 mutations are etiologic in >40% of cases of Rieger syndrome. We demonstrate that the birth prevalence of omphalocele is significantly higher in Rieger syndrome than in the general population, with omphaloceles found in 0.03% in the Iowa newborn population and 4.3% of patients with Rieger syndrome. Our objective was to screen coding and conserved non-coding regions of PITX2 for mutations in 209 patients with omphalocele. We identified remarkable evolutionarily conserved regions by comparing the 3'UTR of Pitx2 in 13 vertebrate and 3 invertebrate species. No mutations changing the amino acid sequence were found within the omphalocele population. In one case of omphalocele with VATER-like additional anomalies, a three nucleotide deletion was found in the 3'UTR. This deletion was not seen in 1,186 controls. Also in the 3'UTR, we identified a single nucleotide polymorphism at a highly conserved residue. Our findings suggest additional studies of PITX2 conserved regions will be valuable. We also screened the omphalocele cases for mutations in exon 5 of the gene FLNA. Mutations in FLNA have been shown to cause a broad range of congenital malformations, including otopalatodigital syndrome type 2 in which a missense mutation occurring in exon 5 of FLNA results in omphalocele as part of the phenotype. We did not find any mutations in exon 5 of FLNA in 179 omphalocele cases studied.

The development of virus-resistant alfalfa, Medicago sativa L.

We have generated more than 100 transgenic alfalfa plants, via Agrobacterium-mediated gene transfer, from genotypes selected from five alfalfa cultivars. These plants express the genes for kanamycin resistance and for the coat protein of alfalfa mosaic virus (AMV). The strongest expressers accumulated nearly 500 ng coat protein per mg soluble leaf protein. AMV inoculation of protoplasts from these strong expressers indicated that they were resistant to infection by AMV, while protoplasts from plants containing about a hundred-fold less coat protein and from control untransformed plants were not. Transgenic alfalfa plants containing large amounts of coat protein were, likewise, resistant to AMV. These plants did not develop systemic infections following inoculation with up to 50 micrograms/ml AMV, while inoculated control plants developed systemic infections following inoculation with as little as 10 micrograms/ml AMV. These results demonstrate that expression of the AMV coat protein gene confers resistance to AMV infection in transgenic alfalfa plants.

Expression of alfalfa mosaic virus RNA 4 in transgenic plants confers virus resistance.

Agrobacterium-mediated transfer from a binary vector was used to produce transgenic Nicotiana tabacum plants that expressed coat protein of the plant virus, alfalfa mosaic virus (AMV). Expression levels of the chimeric gene, which was under the control of the cauliflower mosaic virus 19S promoter, were determined in primary transformed plants, in the progeny from self-fertilization and in the progeny from crosses to normal tobacco. RNA transcripts that were of the expected size as well as a protein of the M(r) and antigenicity of AMV coat protein accumulated in the transgenic plants. Plants that expressed the highest levels of coat protein developed fewer primary infections following inoculation with two strains of AMV and developed systemic infection slower than did plants that did not express coat protein. Resistance was specifically against virions of the AMV strains. AMV RNA and the unrelated virus, tobacco mosaic virus, were as infectious on progeny that expressed coat protein as they were on progeny that did not. The relationship between the virus resistance expressed by these transgenic plants and that observed in virus cross-protection is discussed.

Expression of alfalfa mosaic virus RNA 4 cDNA transcripts in vitro and in vivo.

Copies of alfalfa mosaic virus (AMV) RNA 4 synthesized by transcription in vitro were shown to be biologically active in protoplasts when inoculated together with a mixture of AMV RNAs 1, 2, and 3. A 5' cap was essential for activity, whereas 15 nonviral nucleotides at the 5' end of the RNA transcripts did not detectably affect activity. Transcripts that terminated 65 nucleotides from the 3' end of the coding region and transcripts that lacked all or part of the 3'-noncoding region were effective messengers in a wheat germ cell-free translation system. None of the incomplete transcripts, however, were as biologically active in protoplasts or in Xenopus laevis oocytes as were complete transcripts. This suggests that the 3' end of RNA 4 is involved in translational stability.

Human leukocyte interferon does not inhibit alfalfa mosaic virus in protoplasts or tobacco tissue.

Enzyme-linked immunosorbent assays and local lesion infectivity assays showed that recombinant leukocyte interferons, rIFN-alpha A and rIFN-alpha D, did not reproducibly affect the accumulation of alfalfa mosaic virus in tobacco leaf discs or in tobacco or alfalfa protoplasts when applied within 1 hr after inoculation with virus.