Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes.

DNA was extracted from T5 and BF23 phage carrying amber mutations in genes A2, A1, or D9 and tested for its ability to transfect su minus spheroplasts. DNA from T5 am231, defective in gene A2, transfects Escherichia coli su minus recB minus spheroplasts with an efficiency of 16% of that of wild-type T5 DNA, whereas DNA from T5 am16d or BF23 am57, both defective in gene A1 or its equivalent, transfects E. coli su minus recB minus spheroplasts with an efficiency of 1.4% of that of wild-type T5 DNA, provided E. coli su+ bacteria is used as the indicator in all cases. More than 95% of the progeny from the am231, am16d, and am57 DNA that transfects su minus recB minus spheroplasts is still amber mutant. From these efficiencies of transfection we conclude that the product of gene A2 functions mainly in the mechanism of transfer of phage DNA to intact host cells, and that this function is not essential for transfection of spheroplasts. We also conclude that gene A1 controls functions in addition to DNA transfer, in agreement with previous studies which show that mutations in gene A1 have a pleiotropic effect. Apparently, the absence of these additional functions controlled by gene A1 leads to a high frequency of abortive infection. DNA from amber mutants defective in either gene A1 or A2 does not appreciably transfect su minus rec+ spheroplasts, indicating that the products of these two genes may both be needed to protect T5 DNA from the very active rec BC nuclease in spheroplasts.     

Phe783, Thr797, and Asp804 in transmembrane hairpin M5-M6 of Na+,K+-ATPase play a key role in ouabain binding

Ouabain is a glycoside that binds to and inhibits the action of Na+,K+-ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H+,K+-ATPase and Na+,K+-ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na+,K+-ATPase in a backbone of H+,K+-ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H+,K+-ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na+,K+-ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H+,K+-ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na+,K+-ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na+,K+-ATPase suggests that Asp804, in contrast to Phe783 and Thr797, does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.     

A G43 to U43 mutation in E. coli tRNAtyrsu3+ which affects processing by RNase P.

A novel mutation in the anticodon stem of E. coli tRNA1Tyrsu3+ (G43 to U43) has been characterized. The gene coding for the mutant tRNA, carried by phage phi 80DHA61.3 a derivative of phi 80psu3+su0, produces only 20% of mature suppressor tRNA as compared with phi 80psu3+. Both the mutant tRNA precursor and mature tRNA have an altered conformation. The precursor tRNA coded for by phi 80DHA61.3 is processed by RNase P more slowly than the su3+ precursor and does not form as stable an enzyme-substrate complex as does su3+ precursor. phi 80 DHA61.3 also contains a large deletion which begins in the spacer region between the su3+ gene and the su0- gene, extends through the su0- gene and includes most of the repeated region following the tRNA genes.     

Identification of viral induced genes in Ig+ leucocytes of Japanese flounder Paralichthys olivaceus, by differential hybridisation with subtracted and un-subtracted cDNA probes

Up-regulated genes of leucocytes expressing immunoglobulin (Ig+ leucocytes) of hirame rhabdovirus (HRV)-infected Japanese flounder were identified by differential hybridisation, using subtracted and un-subtracted cDNA probes. Ig+ leucocytes were separated from apparently healthy and HRV-infected Japanese flounder by the magnetic beads antibody method using mouse anti-Japanese flounder Ig monoclonal antibody (mab). A cDNA library was constructed from HRV-infected Japanese flounder leucocytes, and was screened with subtracted cDNA probes enriched in genes up-regulated by HRV infection. Fifty cDNAs were isolated for further analysis. These included cDNAs coding for homologues of interferon-inducible 56K protein (IFI56), Stat3, CEF-10, RGS5, inducible poly(A) binding protein, prolylcarboxylpeptidase, basigin III (Ig superfamily), MUC-18 (Ig superfamily), proteasome-nexin 1 (SERPIN), herpes virus entry mediator (TNFR family), collagenase III, gelatinase-b, megakaryocyte stimulating factor, Rab8-interacting protein, IgM, IgD and 20 unknown cDNA clones. The majority of these identified genes are reported for the first time in fish. From leucocytes mRNA for homologues of IFI56, CEF-10, Stat3, SERPIN and inducible poly (A) binding protein expression was shown to increase following HRV infection.     

Comparative studies of CD3- and CD3+ CD56+ cells: examination of morphology, functions, T cell receptor rearrangement, and pore-forming protein expression.

Both CD3- and CD3+ CD56+ effector cells can mediate non-MHC-restricted lysis in the absence of activation. Previous studies have shown that both of these subsets can be augmented with IL-2. In the present study, we have examined further the phenotypic markers expressed on these cells as well as the functional capacities of these subsets, including LAK activity, cytokine expression, and pore-forming protein (PFP) production. In addition, these populations were analyzed for clonality by Southern blot analysis of the T cell receptor beta chain gene constant region. The CD3-, CD56+ and CD3+, CD56+ lymphocytes were quite similar in their phenotypic markers, although the CD3+, CD56+ lymphocytes lacked high levels of IL-2 receptor beta chain and did not express CD16. The CD3+, CD56+ lymphocytes mediated non-MHC-restricted lysis, but failed to express LAK activity or be induced by IL-2 to secrete IFN gamma, a characteristic of the CD3-, CD56+ lymphocytes. The T cell receptor beta chain gene pattern of the CD3+, CD56+ lymphocytes was characteristic of a polyclonal cell population. Of interest, both populations of cells appeared morphologically to be large granular lymphocytes that contain PFP in their cytoplasmic granules. Therefore these CD56+ subsets provide a new model to study several questions related to non-MHC-restricted target cell lysis, including the identification of novel receptors involved in target cell recognition and/or triggering as well as the biochemical pathways implicated in cellular lysis.     

Suppression of Amber Mutations of Bacteriophage T4 Gene 43 (DNA Polymerase) by Translational Ambiguity

The growth properties of twelve different amber (am) mutants of bacteriophage T4 gene 43 (DNA polymerase) were examined by using nonpermissive (su(-)) as well as permissive (su(+)) Escherichia coli hosts. It was found that most of these mutants were measurably suppressed in su(-) hosts by translational ambiguity (misreading of codons during protein synthesis). The ability of these mutants to grow in response to this form of weak suppression probably means that the T4 gene 43 DNA polymerase can be effective in supporting productive DNA replication when it is supplied in small amounts. By similar criteria, studies with other phage mutants suggested that the products of T4 genes 62 (uncharacterized), 44 (uncharacterized), 42 (dCMP-hydroxymethylase), and 56 (dCTPase) are also effective in small amounts. Some T4 gene products, such as the product of gene 41 (uncharacterized), seem to be partially dispensable for phage growth since am mutants of such genes do propagate, although weakly, in streptomycin-resistant su(-) hosts which appear to have lost the capacity to suppress am mutations by ambiguity.     

Genetic interactions of modifier genes and modifiable alleles in Drosophila melanogaster

We have examined the effects of mutations in the six allele-specific modifier genes su(Hw), e(we), su(f), su(s), su(wa), and su(pr) on the expression of 18 modifiable alleles, situated at 11 loci. Ten of the modifiable alleles are associated with insertions of the gypsy retrotransposon and the others include alleles associated with insertions of copia and 412. We tested or retested 90 of the 108 possible combinations and examined the expression of modifiable alleles in flies mutant for pairs of modifier genes in various heterozygous and homozygous configurations. Our principal findings are: (1) a screen of 40,000 mutagenized X chromosomes yielded three new mutations in known modifier genes, but revealed no new modifier genes; (2) the modification effects of different mutations in a given modifier gene were qualitatively similar; (3) each of the six modifiers suppressed some modifiable alleles, enhanced others, and had no noticeable effect on still others; (4) the modifier genes could be placed in four classes, according to their effects on the gypsy-insertion alleles; and (5) the effects of mutations in different modifier genes combined additively. Implications of these results for models of modifier gene action are discussed.     

Innovation from reduction: gene loss, domain loss and sequence divergence in genome evolution

Analyses of genome sequences have revealed a surprisingly variable distribution of genes, reflecting the generation of novel genes, lateral gene transfer and gene loss. The impact of gene loss on organisms has been difficult to examine, but the loss of protein coding genes, the loss of domains within proteins and the divergence of genes have made surprising contributions to the differences among organisms. This paper reviews surveys of gene loss and divergence in fungal and archaeal genomes that indicate suites of functionally related genes tend to undergo loss and divergence. Instances of fungal gene loss highlighted here suggest that specific cellular systems have changed, such as Ca 2+ biology in Saccharomyces cerevisiae and peroxisome function in Schizosaccharomyces pombe. Analyses of loss and divergence can provide specific predictions regarding protein-protein interactions, and the relationship between networks of protein interactions and loss may form a part of a parametric model of genome evolution.     

Overproduction of the EcoRII endonuclease and methylase by Escherichia coli strains carrying recombinant plasmids constructed in vitro

Recombinant DNA molecules were constructed from the plasmid pIL203 and the EcoRI-fragment of N3 plasmid containing EcoRII endonuclease and methylase genes and also a gene for resistance to sulfanilamide. The pIL203 plasmid, used as a vector, consisted of the Bam HI-EcoRI-fragment of the plasmid pBR322 conferring resistance to ampicillin and the Bam HI-EcoRI-fragment of lambda phage containing promoters, a thermosensitive mutation in the cI gene and a suppressible amber mutation in the cro gene. Ampicillin-sulfanilamide-resistant clones were selected and tested for their restriction and modification phenotype. The recombinant plasmid DNA, isolated from ApRSuR-resistant clones, which restricted and modified phage lambda imm21 with EcoRII specificity, had the EcoRI-fragment with EcoRII genes in a single orientation. The recombinant plasmid pSK323 was transferred into E. coli strains with su-, su1, su2 or su3 phenotypes. The synthesis of products of EcoRII genes by these strains grown at 37 degrees C is increased by 10--50-fold.     

Identification of preferentially expressed mRNAs in retina and cochlea

To search for genes that could be involved in genetic disorders primarily involving the retina and the cochlea, we tried to identify mRNAs preferentially expressed in retina and cochlea and to establish their chromosomal localization. Two approaches were employed. First, a mouse subtracted library (retina + cochlea against liver + brain) was generated. Randomly selected cDNA clones were sequenced and compared to databases. Tissue expression of some of them was analyzed by RT-PCR. Using radiation hybrid cell lines, the mouse chromosomal localization was determined for those showing the highest level in the retina and the cochlea. Second, human Expressed Sequence Tags (ESTs) with preferential expression in the retina and the cochlea were searched for in databases, and chromosomal localization was also established. From 171 sequenced clones, 73 were classified as known genes (with 17 clones coding for 6 genes), 86 were homologous to ESTs, and 12 were unidentified. Of 108 selected clones, 22 (18.5%) had the highest level of expression in the retina and/or the cochlea, while expression was higher in another tissue or ubiquitous for 60 (55.5%) and 22 (20.4%) of them, respectively. By RT-PCR, one clone similar to the mouse Asic3 cDNA (proton-gated channel) was found mainly in the retina and cochlea, but its human ortholog was widely expressed. We selected 17 ESTs from the UniGene database with restricted expression including in the retina and cochlea. We mapped 10 of these ESTs as well as four mouse clones from the subtracted library. Some of them localized to morbid intervals. The combined information from expression analysis and chromosomal localization allowed for the identification of potential candidate genes for retinal diseases (CORD8, CORD9) and syndromic blindness/deafness/renal defects.     

Genomic profiles for human peripheral blood T cells, B cells, natural killer cells, monocytes, and polymorphonuclear cells: comparisons to ischemic stroke, migraine, and Tourette syndrome

Blood genomic profiling has been applied to disorders of the blood and various organ systems including brain to elucidate disease mechanisms and identify surrogate disease markers. Since most studies have not examined specific cell types, we performed a preliminary genomic survey of major blood cell types from normal individuals using microarrays. CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ natural killer cells, and CD14+ monocytes were negatively selected using the RosetteSep antibody cocktail, while polymorphonuclear leukocytes were separated with density gradient media. Genes differentially expressed by each cell type were identified. To demonstrate the potential use of such cell subtype-specific genomic expression data, a number of the major genes previously reported to be regulated in ischemic stroke, migraine, and Tourette syndrome are shown to be associated with distinct cell populations in blood. These specific gene expression, cell-type-related profiles will need to be confirmed in larger data sets and could be used to study these and many other neurological diseases.     

A statistical analysis of nucleotide sequences of introns and exons in human genes

DNA sequences of 56 human genes for which information on both exons and introns was available were examined. The variance in G+C content among genes is estimated and shown to be substantial. There is a high correlation in G+C content between exons and introns within the same gene. The dinucleotide frequencies of introns are similar to those of intergenic spacer regions and are in reasonable agreement with predictions from substitution rates estimated from pseudogenes, except that the observed deficiency of TA doublets is not predicted. Duplicated bases also show a frequency greater than the expectation under independence. There is marked variability among genes in the frequency of the doublet CG relative to its expectation under independence. This variation is evolutionarily conserved and is correlated with the G+C content. Pseudogenes behave as if they are in a low -G+C, CG-deficient part of the genome, although the genes from which they arose are variable in these respects.      

A comparative study of IL-12 (cytotoxic lymphocyte maturation factor)-, IL-2-, and IL-7-induced effects on immunomagnetically purified CD56+ NK cells.

IL-12, or cytotoxic lymphocyte maturation factor, is a recently cloned cytokine shown to influence lymphokine-activated killer cells activity in heterogeneous lymphocyte populations, proliferative activity as a costimulus in PBMC/PBL populations and IFN-gamma production in PBL. We have investigated the effects of IL-12 on immunomagnetically highly purified CD56+ lymphocytes, and compared the effects with those of IL-7 and IL-2. Our results show that IL-12 directly generated high lymphokine-activated killer cell activity in CD56+ NK cells, without the need for accessory cells. The IL-12-induced lymphokine-activated killer cell activity reached 50% of what was obtained with IL-2. In contrast, only low proliferative activity was induced by IL-12, as 10% of the IL-2-induced- and approximately 50% of the IL-7-induced proliferative activity was detected with IL-12. The CD56+ cells expressed high levels of IL-2R alpha and 75-kDa TNFR in response to IL-12, comparable to what was registered with IL-2 and IL-7. Furthermore, an extensive up-regulation of the CD56 Ag, to the level obtained with IL-2, was detected in the CD56+ NK cells in the presence of IL-12. Stimulation with IL-7 resulted in a more limited CD56 up-regulation in the CD56+ NK cells. Low concentrations of TNF-alpha were produced in response to both IL-12 and IL-7, with little or no TNF-beta production. Time course of the IL-2-induced TNF production revealed an initial TNF-alpha production, whereas significant levels of TNF-beta were detected after 72 h. The effects of both IL-12 and IL-7 on the CD56+ NK cells were inhibited by an anti-TNF-alpha mAb. Thus, IL-12 can directly influence NK cell activities in purified CD56+ cells, and endogenously produced TNF-alpha is involved in mediating the effects of both IL-12 and IL-7.     

Search for revertants of the glutamine mischarging mutans of Escherichia coli su+3 tyrosine suppressor tRNA that are able to insert tyrosine at the site of amber mutation.

We have isolated a bacterial amber mutation (nadam) that is suppressed by the tyrosine inserting suppressor su+3 but not by the glutamine (su+2, su+3 A1, su+3 G82 and su+3 A1G82), serine (su+1) and leucine (su+6) inserting suppressors. The su+7 suppressor which inserts glutamine and tryptophan also suppresses this mutation indicating that tryptophan, in addition to tyrosine, is accepted at the site of amber mutation. We have used this amber mutation to search for revertants of the su+3 glutamine mischarging mutants su+3 A1, su+3 G82 and su+3 A1G82 that are able to insert tyrosine at the site of amber mutation. Two types of revertants were found in the case of su+3 A1. One type corresponding to the true revertant A1 leads to G, and the other to the second site revertants C81 leads to U (A1U81). The A1U81 revertant has been shown to insert both glutamine and tyrosine at the site of amber mutation. Only true revertants (G82 leads to A) were obtained when su+3 G82 was analyzed. No revertants were obtained in the case of the su+3 A1G82. These results are discussed in relation to aminoacyl-tRNA recognition.     

Distinct induction patterns and functions of two closely related interferon-inducible human genes, ISG54 and ISG56

Human P54 and P56 proteins are tetratricopeptide proteins that are encoded by two closely related genes, ISG54 and ISG56. These genes are induced strongly but transiently when cells are treated with interferons or double-stranded RNA or infected with a variety of viruses. We observed that, although double-stranded RNA or Sendai virus infection induced the two genes with similar kinetics, their induction kinetics in response to interferon-beta were quite different. The induction kinetics by virus infection were also different between two cell lines. Functionally the two proteins were similar. Like P56, P54 bound to the translation initiation factor eIF3 and inhibited translation. However, unlike P56, P54 bound to both the "e" and the "c" subunits of eIF3. Consequently, P54 inhibited two functions of eIF3. Like P56, it inhibited the ability of eIF3 to stabilize the eIF2 x GTP x Met-tRNA(i) ternary complex. But in addition, it also inhibited the formation of the 48 S pre-initiation complex between the 40 S ribosomal subunit and the 20 S complex composed of eIF3, ternary complex, eIF4F, and mRNA. Thus, although similar in structure, the human P54 and P56 proteins are induced differently and function differently.     

Suppressors of gene 32 am mutants that specifically overproduce P32 (unwinding protein) in bacteriophage T4.

A gene 32 amber (am) mutant, amNG364, fails to grow on Escherichia coli Su3+ high temperatures, suggesting that the tyrosine residue inserted at the am codon by Su3+ leads to a temperature-sensitive gene 32 protein (P32). By plating amNG364 on E. coli Su3+ 45 degrees C, several pseudorevertants were found that proved to contain a suppressor (su) mutant in addition to the original am mutation. Crosses of two of these amNG364su strains to am+ phage indicated that the suppressors themselves are in or close to gene 32. Phage strains carrying either of the two su mutations, without amNG364, grew normally. When cells were infected by these su mutants and the proteins produced were examined by sodium dodecyl sulfate-gel electrophroesis, specific overproduction of P32 was found. Maximum overproduction compared to am+ phage was 6.6-fold for one su mutant and 2.4-fold for the other. Other proteins were produced in normal amounts and in normal time sequence. When amNG364su phage were allowed to infect E. coli S/6/5(Su-), the gene 32 am fragments produced were present at the same derepressed levels as in an infection by amNG364 without a suppressor. The suppressor mutations are interpreted as causing derepression of P32 by altering sites in this autogenously regulated protein involved in template recognition. Previously, specific derepression of gene 32 had only been shown using gene 32 conditional lethal mutants grown under restrictive conditions. We have shown that P32 can also be derepressed under permissive conditions, indicating that loss of P32 function is not necessary for specific derepression.     

Conditional lethal mutants of bacteriophage T4 unable to grow on a streptomycin resistant mutant of Escherichia coli.

Sixteen conditional lethal mutants of bacteriophage T4D have been isolated which grow on Escherichia coli CR63 (a su+ streptomycin-sensitive K12 strain) but are restricted by CR/s (a streptomycin-resistant derivative of CR63). These mutants have been given the prefix str. Four of these mutants are amber and 12 appear to be missense. Eleven of the 12 missense mutants appear to be "pseudo-amber" (i.e. they are restricted by a su- E. coli B strain but not by a su- K12 strain); the other missense mutant was not restricted by either B or K12. The str mutations mapped in 12 different genes. Most were clustered in a region of early genes (gene 56 to gene 47). Fifty-eight amber and 10 "pseudo-amber" mutants isolated previously for their inability to grow on E. coli B were tested for restriction by CR/s. All the amber mutants grew normally on CR/s, whereas all 10 "pseudo-amber" mutants were restricted by CR/s. This implies that the phenotype of the "pseudo-amber" mutants is the result of a ribosomal difference between the permissive host CR63 and the restrictive hosts B and CR/s. These str mutants should prove to be useful alternatives to amber mutants for genetic and biochemical studies of bacteriophage T4 and for studies of the E. coli ribosome. It should be possible ot isolate similar mutants in other bacteriophages provided that streptomycin resistant hosts are available.