Insulin-like growth factor 1 and a cytosolic tyrosine kinase activate chloride outward transport during maturation of hippocampal neurons.

The development of hyperpolarizing inhibition is an important step in the maturation of neuronal networks. Hyperpolarizing inhibition requires Cl(-) outward transport that is accomplished by KCC2, a K(+)/Cl(-) cotransporter. We show that cultured hippocampal neurons initially contain an inactive form of the KCC2 protein, which becomes activated during subsequent maturation of the neurons. We also show that this process is accelerated by transient stimulation of IGF-1 receptors. Because the transporter can be rapidly activated by coapplication of IGF-1 and an Src kinase and can be deactivated by membrane-permeable protein tyrosine kinase inhibitors, we suggest that activation of K(+)/Cl(-) cotransporter function by endogenous protein tyrosine kinases mediates the developmental switch of GABAergic responses to hyperpolarizing inhibition.

Impaired electrical signaling disrupts gamma frequency oscillations in connexin 36-deficient mice.

Neural processing occurs in parallel in distant cortical areas even for simple perceptual tasks. Associated cognitive binding is believed to occur through the interareal synchronization of rhythmic activity in the gamma (30-80 Hz) range. Such oscillations arise as an emergent property of the neuronal network and require conventional chemical neurotransmission. To test the potential role of gap junction-mediated electrical signaling in this network property, we generated mice lacking connexin 36, the major neuronal connexin. Here we show that the loss of this protein disrupts gamma frequency network oscillations in vitro but leaves high frequency (150 Hz) rhythms, which may involve gap junctions between principal cells (Schmitz et al., 2001), unaffected. Thus, specific connexins differentially deployed throughout cortical networks are likely to regulate different functional aspects of neuronal information processing in the mature brain.

Mice with a targeted intronic deletion in the Col1a1 gene respond to bleomycin-induced pulmonary fibrosis with increased expression of the mutant allele.

Experiments designed to examine the role of the first intron in regulation of the Col1a1 gene by transfection and in transgenic mice have led to conflicting conclusions. Recently, Hormuzdi et al. [Hormuzdi, S.G., Penttinen, R., Jaenisch, R., Bornstein, P., 1998. A gene-targeting approach identifies a function for the first intron in expression of the alpha1(I) collagen. Mol. Cell. Biol. 18, 3368-3375.] created a targeted deletion in this intron in mice and demonstrated an age-dependent reduction in expression of the mutated allele in lung and skeletal muscle. In this study, intratracheal instillation of bleomycin in mice was used to induce pulmonary fibrosis in control and intron-deleted animals. This stimulus for collagen synthesis was associated with a marked upregulation of the intron-deleted allele in mutant mice. Our results establish that the inhibition of expression of the mutant Col1a1 gene is not fixed, since the gene can still respond to physiological signals. We propose that cis-acting elements, elsewhere in the gene, can compensate for the lack of intronic sequences in the mutated Col1a1 allele and account for the conditional nature of the inhibition. This model has the potential to resolve the conflicting results of previous transfection and transgenic experiments in which different fragments of the Col1a1 gene were used.

A gene-targeting approach identifies a function for the first intron in expression of the alpha1(I) collagen gene.

The role of the first intron of the Col1A1 gene in the regulation of type I collagen synthesis remains uncertain and controversial despite numerous studies that have made use of transgenic and transfection experiments. To examine the importance of the first intron in regulation of the gene, we have used the double-replacement method of gene targeting to introduce, by homologous recombination in embryonic stem (ES) cells, a mutated Col1A1 allele (Col-IntDelta). The Col-IntDelta allele contains a 1. 3-kb deletion within intron I and is also marked by the introduction of a silent mutation that created an XhoI restriction site in exon 7. Targeted mice were generated from two independently derived ES cell clones. Mice carrying two copies of the mutated gene were born in the expected Mendelian ratio, developed normally, and showed no apparent abnormalities. We used heterozygous mice to determine whether expression of the mutated allele differs from that of the normal allele. For this purpose, we developed a reverse transcription-PCR assay which takes advantage of the XhoI polymorphism in exon 7. Our results indicate that in the skin, and in cultured cells derived from the skin, the intron plays little or no role in constitutive expression of collagen I. However, in the lungs of young mice, the mutated allele was expressed at about 75% of the level of the normal allele, and in the adult lung expression was decreased to less than 50%. These results were confirmed by RNase protection assays which demonstrated a two- to threefold decrease in Col1A1 mRNA in lungs of homozygous mutant mice. Surprisingly, in cultured cells derived from the lung, the mutated allele was expressed at a level similar to that of the wild-type allele. Our results also indicated an age-dependent requirement for the intact intron in expression of the Col1A1 gene in muscle. Since the intron is spliced normally, and since the mutant allele is expressed as well as the wild-type allele in the skin, reduced mRNA stability is unlikely to contribute to the reduction in transcript levels. We conclude that the first intron of the Col1A1 gene plays a tissue-specific and developmentally regulated role in transcriptional regulation of the gene. Our experiments demonstrate the utility of gene-targeting techniques that produce subtle mutations for studies of cis-acting elements in gene regulation.

Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission.

The ability to contribute to the germ line is the most important experimental feature of embryonic stem (ES) cells. Using ES cells, it is possible to introduce targeted mutations into any gene and to derive the corresponding mutant mice. A common problem with this technology is that the ES cells often lack or have only a low efficiency of germ line transmission. To address this issue, we examined the relationship between the growth rate and karyotype of ES cells, and their ability to contribute to the germ line. We found that chromosomal abnormalities occurred rather frequently in ES cells. Cells having an abnormal number of chromosomes, in particular trisomy 8, were found in three independently derived ES cell lines, and this abnormality conferred a selective growth advantage on these cells. Selection of abnormal cells led to depletion and eventual loss of normal ES cells during consecutive passages. In comparison with parental ES cells, ES cells with trisomy 8 contributed rarely to the germ line. This realization allowed us to select, based upon ES cell clone morphology, those clones with the highest probability of contributing to the germ line. This insight is of practical value for any given gene targeting experiment as it permits optimization of the rate of success without having to rely on more elaborate tests such as karyotyping individual clones prior to blastocyst injection.

Genetic analysis of beet curly top virus: examination of the roles of L2 and L3 genes in viral pathogenesis.

The monopartite DNA genome of beet curly top geminivirus (BCTV, strain Logan) contains four leftward (complementary sense) open reading frames (ORFs) designated L1, L2, L3, and L4. We investigated the functions of the L2 and L3 ORFs by mutational analysis. We found that in Nicotiana benthamiana and sugarbeet plants, neither a functional L2 nor a functional L3 gene is required for infectivity. Double mutants were also infectious, and no evidence for a synergistic effect of these genes was evident. However, while sugarbeet plants inoculated with L2 or L3 mutants showed symptoms that were indistinguishable from those elicited by wild type virus, mutant-inoculated N. benthamiana plants displayed a novel phenotype in which recovery of the plant from initially severe disease symptoms was greatly enhanced. Enhanced recovery was associated with a large reduction in viral DNA levels. Our studies did not provide evidence for functional homology between the BCTV L2 gene and its presumed homologue (AL2) in the bipartite geminiviruses. In contrast, mutants with lesions in the L3 ORF accumulated three- to five-fold less DNA than wild type virus in a protoplast replication assay, consistent with the interpretation that the BCTV L3 gene is a homologue of the bipartite geminivirus AL3 gene which is known to function as a replication enhancer. Functional homology was directly confirmed in experiments which demonstrated that the BCTV L3 gene can complement a tomato golden mosaic virus AL3 mutant, and vice versa.

Genetic analysis of beet curly top virus: evidence for three virion sense genes involved in movement and regulation of single- and double-stranded DNA levels.

The monopartite DNA genome of beet curly top geminivirus (BCTV, strain Logan) contains four leftward, complementary sense open reading frames (ORFs) designated L1, L2, L3, and L4 and three rightward, virion sense ORFs designated R1, R2, and R3 (R1 encodes the coat protein). The R3 ORF has not been reported previously in the BCTV genome, and evidence for three functional virion sense genes on one genome component has not been presented before for any geminivirus. We investigated the functions of the virion sense ORFs by introducing mutations into each of them. We found that in Nicotiana benthamiana plants, BCTV genomes containing mutations in ORF R1 were not infectious, whereas an R3- mutant was very weakly infectious. The small proportion of plants infected by the R3- mutant remained asymptomatic and contained greatly reduced amounts of viral DNA. An R2- mutant was highly infectious but asymptomatic, and in infected plants it accumulated mostly the double-stranded DNA (dsDNA) replicative form in nearly wild-type amounts. All of the mutants replicated in tobacco protoplasts, although R1- and R2- mutants accumulated reduced amounts of genomic single-stranded DNA (ssDNA) relative to wild-type virus. In the case of R2- mutants, the reduction was large (approx. ninefold) and was accompanied by a similar increase in dsDNA levels. The results suggest that the R1 and R3 gene products are required for efficient movement of the virus in the infected plant, whereas the R2 gene product may be involved in the regulation of ssDNA vs dsDNA levels.

A number of subgenomic DNAs are produced following agroinoculation of plants with beet curly top virus.

In addition to ss and ds genomic DNA, agroinoculation of Nicotiana benthamiana plants with the Logan strain of the geminivirus beet curly top virus (BCTV) consistently resulted in de novo production of subgenomic DNAs on initial passage. Single-stranded and dsDNA forms representing at least seven size classes (0.8 to 1.8 kb) of subgenomic DNA were observed in total DNA extracts from inoculated plants. Extracts from infected sugar beet and tomato contained variable but usually smaller amounts of subgenomic DNAs, suggesting that their production may be influenced by the host species. Restriction endonuclease mapping and partial nucleotide sequencing of three independent clones of a 1.5 kb size class indicated that this subgenomic DNA is produced from the standard viral genome by two separate deletion events. One deletion of 941 bp includes portions of the leftward open reading frames (ORFs) L1, L2 and L3, while the other deletion of 579 bp encompasses portions of the intergenic region and the rightward ORFs R1, R2 and R3. The data indicate that the 1.5 kb BCTV subgenomic DNA is a defective DNA that has retained cis-elements essential for replication.

Genetic analysis of tomato golden mosaic virus: ORF AL2 is required for coat protein accumulation while ORF AL3 is necessary for efficient DNA replication.

Tomato golden mosaic virus (TGMV) is a geminivirus whose genome is divided between two DNA components, designated A and B. The TGMV genome contains six open reading frames (ORFs) which can encode proteins of greater than 10 kDa. We have used a protoplast transfection system to determine the effects of viral proteins, as defined by these ORFs, on the accumulation of viral DNA in infected cells. The accumulation of cost protein was also examined in leaf discs. Our results indicate that mutations in ORFs AR1 and AL2 do not affect viral double-stranded DNA (dsDNA) levels, although AR1 and AL2 mutants accumulate only small amounts of single-stranded viral DNA (ssDNA). In contrast, a large reduction in both ss- and dsDNA levels is observed when a mutation is introduced into ORF AL3. Mutations within either of the two DNA B ORFs do not affect DNA replication. The AL3, BR1, and BL1 mutants are capable of synthesizing coat protein; however, coat protein is not detected in leaf discs inoculated with AR1 or AL2 mutants. Testable models are proposed to explain the influence of AL2 protein on coat protein accumulation and to account for the stimulation of viral DNA synthesis mediated by the AL3 gene product.