Isolation of bacteriophage T4 baseplate proteins P7 and P8 and in vitro formation of the P10/P7/P8 assembly intermediate.

Two bacteriophage T4 proteins, P7 and P8, which are components of the phage baseplate have been purified to apparent homogeneity. P7 and P8 are the protein products of T4 genes 7 and 8. A plasmid has been constructed which contains approximately 5 kilobases of T4 DNA, including genes 7 and 8, under the control of the tac promoter. Induction of Escherichia coli W3110iQ cells containing this plasmid resulted in the production of functional P7 and P8. Standard protein isolation procedures were used to purify both P7 and P8 from extracts of induced cells. In T4-infected cells, these two proteins and P10 interact in a strictly ordered sequential manner (P10 + P7----P10/P7,P10/P7 + P8----P10/P7/P8) to form an intermediate in the baseplate assembly pathway. The three purified proteins assembled in vitro to form a limited number of oligomeric species, as determined by nondenaturing gel electrophoresis. P10 and P7 interacted in vitro to form two assemblies with distinct electrophoretic mobilities, both containing P10 and P7. Addition of P8 to this mixture resulted in the disappearance of both P10/P7 species and the appearance of a single new assembly with a different electrophoretic mobility. These interactions occurred without the addition of any catalyst or cofactors. Isolated P11 appeared to add as predicted to the in vitro-formed complexes without affecting the formation of the two P10/P7 or the single P10/P7/P8 intermediates. Interactions between P7 and P8 in the absence of P10 or interactions between P10 and P8 in the absence of P7 could not be detected. These data indicate that purified P10, P7, and P8 interact in vitro in a manner completely in accord with the published assembly pathway and thus establish a system for further study of the regulation of the formation of this assembly intermediate in vitro.     

Intron definition in splicing of small Drosophila introns.

Approximately half of the introns in Drosophila melanogaster are too small to function in a vertebrate and often lack the pyrimidine tract associated with vertebrate 3' splice sites. Here, we report the splicing and spliceosome assembly properties of two such introns: one with a pyrimidine-poor 3' splice site and one with a pyrimidine-rich 3' splice site. The pyrimidine-poor intron was absolutely dependent on its small size for in vivo and in vitro splicing and assembly. As such, it had properties reminiscent of those of yeast introns. The pyrimidine-rich intron had properties intermediate between those of yeasts and vertebrates. This 3' splice site directed assembly of ATP-dependent complexes when present as either an intron or exon and supported low levels of in vivo splicing of a moderate-length intron. We propose that splice sites can be recognized as pairs across either exons or introns, depending on which distance is shorter, and that a pyrimidine-rich region upstream of the 3' splice site facilitates the exon mode.     

Efficient selection of 3'-terminal exons from vertebrate DNA.

Identification of expressed sequences within genomic DNA is a hurdle in the characterization of complex genomes. We developed an exon trapping scheme that provides a positive selection for vertebrate 3'-terminal exons. A copy of the trapped exon sequence is obtained by RT/PCR amplification. The technique detects valid terminal exons without interference from partial exons or non-specific sequences, including simple human repeated sequences. Application to random human cosmids yielded one unique trapped terminal exon per cosmid on average. Because vertebrate terminal exons average 600-700 nucleotides in length, the technique provides transcribed sequences of sufficient length to assist further mapping efforts.     

An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon.

The average length of a vertebrate axon is approximately 130 nt. Decreasing the size of an internal axon to less than 51 nt induces axon skipping, implying a minimal size for exons. A few constitutively included internal exons, however, are extremely small. To investigate if such micro-exons require special mechanisms for their inclusion, we studied the sequences necessary for inclusion of a 6-nt axon from chicken cardiac troponin T (cTNT). In vivo, the cTNT micro-exon was not included in mRNA unless accompanied by a 134-nt sequence located next to the micro-exon in the downstream intron. Increasing the length of the micro-exon alleviated the requirement for the intron element, indicating that the lack of inclusion of the micro-exon in the absence of a facilitating sequence was due to its small size, rather than suboptimal splice sites. The intron element contained six copies of a G-rich 7-nt sequence. Multimers of the repeat supported exon inclusion, indicating that the repeat sequence is an important part of the intron element. The entire intron element activated inclusion of a heterologous 7-nt exon, suggesting that the intron element is a general enhancer for the splicing of micro-exons. In vitro, the intron element and the repeated sequence facilitated splicing of a heterologous exon. Because of the ability of the cTNT intron element to facilitate the splicing of heterologous exons, we have termed the element an intron splicing enhancer (ISE). Interestingly, the ISE demonstrated position independence in that it facilitated inclusion of the heterologous micro-exon when placed either upstream or downstream of the micro-exon. In vitro, the ISE or copies of the ISE G-rich repeat stimulated splicing of an adjacent intron. The ISE thus becomes one of only a few characterized ISEs containing a G-rich repeat and the first to work both upstream and downstream of a target axon.     

Disruption of the Cytokeratin Cytoskeleton and Inhibition of Hepatocytic Autophagy by Okadaic Acid

To learn whether autophagy might be dependent on any of the major cytoskeletal elements, the effect of various cytoskeleton inhibitors on autophagy and cytoskeletal organization was studied in isolated rat hepatocytes. Autophagy, measured as the sequestration of endogenous lactate dehydrogenase, was completely inhibited in isolated rat hepatocytes by the protein phosphatase inhibitor okadaic acid (30 nM). Only small effects were seen with vinblastine (10 μM) or cytochalasin D (10 μM). Indirect immunofluorescence microscopy with antibody to a 55-kDa cytokeratin, corresponding to human cytokeratin 8 (CK8), revealed that whereas control cells contained a well-organized network of cytokeratin intermediate filaments, okadaic acid disrupted this network into small spherical aggregates. Treatment with cytochalasin D or vinblastine, which disrupt microfilaments and microtubules, respectively, had no detectable effect on the cytokeratin filament distribution. Neither the microtubule network (detected by indirect immunofluorescence with antibodies against α- and β-tubulin) nor the actin microfilament network (detected by rhodamine-palloidin) was disrupted by okadaic acid. Naringin (100 μM), a putative protein kinase-inhibitory flavonoid, offered complete protection against the autophagy-inhibitory and cytokeratin-disruptive effects of okadaic acid. Two other flavonoids, genistein (100 μM) and prunin (100 μM) as well as KN-62 (10 μM), a specific inhibitor of Ca²⁺/calmodulin-dependent kinase II), likewise displayed a good ability to protect against the effect of okadaic acid upon cytokeratin organization, while no such protection was seen with H-89 (20 μM), an inhibitor of the cyclic nucleotide-dependent protein kinases, or with H-7 (100 μM), which in addition inhibits protein kinase C. The results suggest that the cytokeratin cytoskeleton of hepatocytes is subject to rapid control by phosphorylation and dephosphorylation and that cytokeratin filaments may somehow be involved in the autophagic process.     

Intragenic Suppression of a Capsid Assembly-Defective P22 Tailspike Mutation

The tailspike protein of bacteriophage P22 assembles with mature capsids during the final reaction in phage morphogenesis. The gene 9 mutation hmH3034 synthesizes a tailspike protein with a change at amino acid 100 from Asp to Asn. This mutant form of trimeric tailspike protein fails to assemble with capsids in vivo. By using in vitro quantitative tailspike-capsid assembly assays, this mutant tailspike trimer can be shown to assemble with capsids at very high tailspike concentrations. From these assays, we estimate that this single missense mutation decreases by 100-500-fold the affinity of the tailspike for capsids. Furthermore, hmH3034 tailspike protein has a structural defect which makes the mature tailspike trimers sensitive to SDS at room temperature and causes the trimers to "partially unfold." Spontaneously arising intragenic suppressors of the capsid assembly defect have been isolated. All of these suppressors are changes at amino acid 13 of the tailspike protein, which substitute His, Leu or Ser for the wild type amino acid Arg. These hmH3034/sup3034 mutants and the separated sup3034 mutants form fully functional tailspike proteins with assembly activities indistinguishable from wild type while retaining the SDS-sensitive structural defect. From the analysis of the hmH3034 mutant and its suppressors, we propose that in the wild-type tailspike protein, the Asp residue at position 100 and the Arg residue at position 13 form an intrachain or interchain salt bridge which stabilizes the amino terminus of the tailspike protein and that the unneutralized positive charge at amino acid 13 in the hmH3034 protein is the cause of the assembly defect of this protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and function of enzymes acting on chitin and chitosan

Enzymatic conversions of chitin and its soluble, partially deacetylated derivative chitosan are of great interest. Firstly, chitin metabolism is an important process in fungi, insects and crustaceans. Secondly, such enzymatic conversions may be used to transform an abundant biomass to useful products such as bioactive chito-oligosaccharides. Enzymes acting on chitin and chitosan are abundant in nature. Here we review current knowledge on the structure and function of enzymes involved in the conversion of these polymeric substrates: chitinases (glycoside hydrolase families 18 & 19), chitosanases (glycoside hydrolase families 8, 46, 75 & 80) and chitin deacetylases (carbohydrate esterase family 4).