Refining the Ciona intestinalis Model of Central Nervous System Regeneration

Background

New, practical models of central nervous system regeneration are required and should provide molecular tools and resources. We focus here on the tunicate Ciona intestinalis, which has the capacity to regenerate nerves and a complete adult central nervous system, a capacity unusual in the chordate phylum. We investigated the timing and sequence of events during nervous system regeneration in this organism.

Methodology/Principal Findings

We developed techniques for reproducible ablations and for imaging live cellular events in tissue explants. Based on live observations of more than 100 regenerating animals, we subdivided the regeneration process into four stages. Regeneration was functional, as shown by the sequential recovery of reflexes that established new criteria for defining regeneration rates. We used transgenic animals and labeled nucleotide analogs to describe in detail the early cellular events at the tip of the regenerating nerves and the first appearance of the new adult ganglion anlage.

Conclusions/Significance

The rate of regeneration was found to be negatively correlated with adult size. New neural structures were derived from the anterior and posterior nerve endings. A blastemal structure was implicated in the formation of new neural cells. This work demonstrates that Ciona intestinalis is as a useful system for studies on regeneration of the brain, brain-associated organs and nerves.     

The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway.

The constitutive transport elements (CTEs) of type D retroviruses are cis-acting elements that promote nuclear export of incompletely spliced mRNAs. Unlike the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1), CTEs depend entirely on factors encoded by the host cell genome. We show that an RNA comprised almost entirely of the CTE of Mason-Pfizer monkey virus (CTE RNA) is exported efficiently from Xenopus oocyte nuclei. The CTE RNA and an RNA containing the RRE of HIV-1 (plus Rev) have little effect on export of one another, demonstrating differences in host cell requirements of these two viral mRNA export pathways. Surprisingly, even very low amounts of CTE RNA block export of normal mRNAs, apparently through the sequestration of cellular mRNA export factors. Export of a CTE-containing lariat occurs when wild-type CTE, but not a mutant form, is inserted into the pre-mRNA. The CTE has two symmetric structures, either of which supports export and the titration of mRNA export factors, but both of which are required for maximal inhibition of mRNA export. Two host proteins bind specifically to the CTE but not to non-functional variants, making these proteins candidates for the sequestered mRNA export factors.     

Differing responses of Nijmegen breakage syndrome and ataxia telangiectasia cells to ionizing radiation

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder. Originally thought to be a variant of ataxia telangiectasia (AT), the cellular phenotype of NBS has been described as almost indistinguishable from that of AT. Since the gene involved in NBS has been cloned and its functions studied, we sought to further characterize its cellular phenotype by examining the response of density-inhibited, confluent cultures of human diploid fibroblasts to irradiation in the G(0)/G(1) phase of the cell cycle. Both NBS and AT cells were markedly sensitive to the cytotoxic effects of radiation. NBS cells, however, were proficient in recovery from potentially lethal damage and exhibited a pronounced radiation-induced G(1)-phase arrest. Irradiated AT cells showed no potentially lethal damage and no G(1)-phase arrest. Both cell types were hypersensitive to the induction of chromosomal aberrations, whereas the distribution of aberrations in irradiated NBS cells was similar to that of normal controls, AT cells showed a high frequency of chromatid-type aberrations. TP53 and CDKN1A (also known as p21(Waf1)) expression was attenuated in irradiated NBS cells, but maximal induction occurred 2 h postirradiation, as was observed in normal controls. The similarities and differences in cellular phenotype between irradiated NBS and AT cells are discussed in terms of the functional properties of the signaling pathways downstream of AT involving the NBS1 and TP53 proteins.     

Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome

The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.     

Biochemical properties of the bromodeoxyuridine-induced guinea pig virus.

The biophysical and biochemical properties of the virus particles released by guinea pig embryo cells treated with 5-bromo-2'-deoxyuridine (BUdR) have been compared to those of the B-type mouse mammary tumor virus (MMTV) and the C-type Rauscher murine leukemia virus. The high-molecular-weight (60 to 70S) RNA of the BUdR-induced guinea pig virus (GPV) has a molecular weight of 8 X 106 when measred by mixed agarose polyacylamide gel electrophoresis. The virus particles isolated from the tissue culture medium of BUdR-induced guniea pig cells have the following properties in common with MMTV: (i) a buoyant density of 1.18 g/ml in sucrose and 1.21 g/ml in CsCl, and (ii) a DNA polymerase that prefers Mg2+ over Mn2+ in an assay using the synthetic template poly(rC):oligo(dG). No nucleic acid sequence homology between GPV RNA and the viral RNAs of the MMTV, murine leukemia virus, hamster sarcoma virus, or Mason-Pfizer monkey virus could be observed in a competition hybridization assay using the radioactive-labeled GPV 60 to 70S RNA. By this same competition by hybridization assay the frequency of GPV proviral sequences was estimated to be at least 83 per haploid cellular genome of guniea pig cells. No nucleic acid sequences related to be GPV RNA were detected in the DNA of normal tissues of mice, rats, cats, dogs, baboons, or humans by direct RNA-DNA hybridization using radioactive GPV60 to 70S RNA.     

Point mutations in the middle of 16S ribosomal RNA of E. coli produced by deletion loop mutagenesis

Using the plasmid pKK3535 , which contains the rrnB operon of Escherichia coli in pBR322, a deletion mutation was constructed which lacks bases 822 to 874 in the middle of the 16S ribosomal RNA. This results in an "amputation" of a very distinct stem and loop structure in the RNA. By forming a heteroduplex between the deletion plasmid and the original pKK3535 and by modifying the single-stranded deletion loops with bisulfite, we produced plasmids containing one or two base changes at positions 839, 840, 841, 867 or 876. The clustering of the mutations near the top of the stem, and the inability to get base changes at other positions, suggests that single alterations at particular positions severely affect the formation of a functional ribosome. The ability to recover mutations at these positions is not determined by the secondary structure of the DNA during bisulfite mutagenesis. Restriction enzyme analysis of 12 revertants from a slow growing mutant (altered at positions 839 and 876) shows that they did not compensate for the mutation by re-establishing the original wild type sequence.     

Oligonucleotide directed mutagenesis of Escherichia coli 5S ribosomal RNA: construction of mutant and structural analysis.

The ribosomal 5S RNA gene from the rrnB operon of E. coli was mutagenised in vitro using a synthetic oligonucleotide hybridised to M13 ssDNA containing that gene. The oligonucleotide corresponded to the 5S RNA sequence positions 34 to 51 and changed the guanosine at position 41 to a cytidine. The DNA containing the desired mutation was identified by dot blot hybridisation and introduced back into the plasmid pKK 3535 which contains the total rrnB operon in pBR 322. Plasmid coded 5S rRNA was selectively labeled with 32p using a modified maxi-cell system, and the replacement of guanosine G41 by cytidine was confirmed by RNA sequencing. The growth of cells containing mutant 5S rRNA was not altered by the base change, and the 5S rRNA was processed and incorporated into 50S ribosomal subunits and 70S ribosomes. The structure of wildtype and mutant 5S rRNA was compared by chemical modification of accessible guanosines with kethoxal and limited enzymatic digestion using RNase T1 and nuclease S1. These results showed that the wildtype and mutant 5S rRNA do not differ significantly in their structure. Furthermore, the formation, interconversion and stability of the two 5S rRNA A- and B-conformers are unchanged.     

Extending ribosomal protein identifications to unsequenced bacterial strains using matrix-assisted laser desorption/ionization mass spectrometry

A protocol has been developed that allows protein identifications using available DNA-based or protein sequences from a reference strain of a bacterial species to be extended to bacterial strains for which no prior DNA-based or protein sequence information exists. The protocol is predicated on careful isolation of a specific sub-cellular group of proteins. In this study, ribosomal proteins were chosen due to their high relative abundance and similarity in copy number per cell. After isolation of ribosomal proteins, MALDI-MS is used to acquire accurate protein molecular weights. An iterative comparison of reference protein molecular weights and identities is made to the resulting data, allowing for the straightforward identification of ribosomal proteins from any non-reference strains. This approach can reveal differences between proteins at the amino acid or post-translational level. The protocol was developed, validated and applied to ribosomal proteins from three strains of the extreme thermophile Thermus thermophilus. This approach revealed that nearly 60% of the ribosomal proteins from all three strains are identical. The extension of protein identification to additional bacterial strains can be useful in phylogenetic studies as well as in biomarker identification.     

Decreased requirement for 4.5S RNA in 16S and 23S rRNA mutants of Escherichia coli

4.5S RNA is the bacterial homolog of the mammalian signal recognition particle (SRP) RNA that targets ribosome-bound nascent peptides to the endoplasmic reticulum. To explore the interaction of bacterial SRP with the ribosome, we have isolated rRNA suppressor mutations in Escherichia coli that decrease the requirement for 4.5S RNA. Mutations at C732 in 16S rRNA and at A1668 and G1423 in 23S rRNA altered the cellular responses to decreases in both Ffh (the bacterial homolog of SRP54) and 4.5S RNA levels, while the C1066U mutation in 16S rRNA and G424A mutation in 23S rRNA affected the requirement for 4.5S RNA only. These data are consistent with a dual role for 4.5S RNA, one involving co-translational protein secretion by a 4.5S-Ffh complex, the other involving free 4.5S RNA.