Development of a system useful for studying the formation of unstable alleles of IS2

Summary IS2-induced deletions of the gal control region were isolated in a plasmid carrying gal OP-308 :: IS2-7. This contains a 54 basepair long, unstable mini insertion within IS2, thus allowing constitutive expression of the gal structural genes. Deletion PPI is 11.9 kilobasepairs (kb) long and is Gal⁺ because it has retained the mini insertion. In PP4 7.2 kb DNA material including markers gal OP, chlD and pgl are deleted. PP4 has lost the mini insertion and is therefore Gal negative. DNA sequencing of the newly formed junction in PP4 reveals that the deletion terminates precisely at nucleotide 1 of IS2 and that no DNA sequence homology is involved in this IS2-mediated deletion formation. PPI segregates Gal^- clones due to the loss of the mini insertion. One such segregant PPIS and PP4 both give only constitutive Gal⁺ revertants, which consist of the previously known mini insertions and also a new class of supermini inserts within IS2 of about 10 to 20 basepairs long. Therefore, PPIS and PP4 can be used to study various parameters involved in the formation of mini insertions.     

Nucleotide sequence of the transposable DNA-element IS2.

The complete sequence of the transposable DNA element IS2 in gal OP-308:: IS2 (I) has been determined. This element is 1.327 bp long. The integrated element is flanked by a five base pair long sequence duplication. The termini of IS2 are not perfect inverted repeats, but a close approximation.     

Analysis of relative reversion frequencies for IS2 insertion mutations in the regulatory region of the galOPETK operon of Escherichia coli

Two previously characterized mutations in the galOPETK operon of Escherichia coli, galOP-3 and galOPE-490, contain IS2 insertions only 1 bp apart in the gal regulatory region; yet only the former yields Gal+ phenotypic revertants at a detectable frequency. We have shown that the galOPE-490 allele comprises two mutations--an IS2(I) insertion at bp+(2-6) (relative to the gal mRNA start site) plus a C/G to A/T transversion at bp + 59. The latter creates an ochre stop codon and lies within the internal site of the bipartite gal operator; it acts as an operator mutation in an in vivo repressor titration assay. Analysis of a newly isolated allele (galOP-490*) which retains the IS2 of galOPE-490 but is galE+ reveals a reversion frequency approximately 30-fold higher than that of galOP-3. Reversion of galOPE-490 is at least 10,000-fold lower and has not been detectable even under conditions conducive to enhanced double mutations in other systems.     

Suppression of polarity of insertion mutations in the gal operon and N mutations in bacteriophage lambda.

Bacterial mutations (psuA and psu) known for their ability to suppress the polarity on nonsense mutations are shown to suppress the polarity of certain insertion mutations in the gal operon. The short insertion, IS1 (800 nucleotide pairs), is about 15 to 50% suppressed, whereas longer insertions, IS2 (1,400 nucleotide pairs), and IS3 (1,200 nucleotide pairs), are not. Some of the polarity suppressor mutations (psu-1, psu-2, and psu-3) are at least partially permissive for N-gene mutations (N7 and N53) of bacteriophage lambda, suggesting a relationship between natural and mutational polar signals. That this relationship may be complex is indicated by the fact that other suppressor mutations, effective in suppressing nonsense or insertion polarity, fail entirely to permit the growth of lambda N mutants.     

IS1 insertion generates duplication of a nine base pair sequence at its target site.

Three independent integrations of the E. coli insertion sequence, IS1, into the gal operon have been analyzed. DNA sequences of portions of the wild-type galT gene which act as the target sites for these insertions, as well as the corresponding gal/IS1 junctions, are reported. Two features are particularly noteworthy. First, similar sequences appearing in inverted orientation consitute the ends of IS1: 18 of the terminal 23 base pairs at each end are identical. Second, in all three insertions, a 9 base pair segment found once in the wild-type sequence at the site of insertion is duplicated and appears in the same orientation at each end of the inserted element. The sequence of this 9 base pair repeat is different for each insertion analyzed. No homology between the inverted repeat sequences at the ends of IS1 and the sequences of the target sites is observed. Models for the mechanism of IS1 insertion are proposed.     

The biochemical and genetic basis for high frequency thiomethyl galactoside resistance in lambda,lambdadg lysogens of Escherichia coli.

In a culture of Escherichia coli K12 gal (lambdadg), cells which form large colonies on agar plates containing galactose and thiomethyl beta-D-galactoside (TMG) appear at high frequency. These clones are resistant to growth inhibition by TMG on galactose minimal medium. Biochemical studies of the steady-state levels of galactokinase and UDPgalactose 4-epimerase suggest that the resistant clones have extra copies of the genes for the galactose-metabolizing enzymes. The mutation for TMG resistance is not located in either the bacterial or the bacteriophage genome, but is probably due to an aberrant association between cell and prophage DNA. Mapping the TMG-resistant characteristic by phage P1 indicates that TMG-resistant bacteria posses at least two GAL+ OPERONS, ONE OF WHICH IS COTRANSDUCIBLe with bio+. In addition, TMG-resistant bacteria behave like lambdadg polylysogens when challenged with the phage lambdaI90c17. From these genetic experiments we conclude that TMG-resistant bacteria arise by duplication of the lambdadg prophage. Finally, gal+ bacteria which carry a single, additional, lambdadg prophage are TMG-resistant. TMG resistance is probably a gal+ gene dosage effect.     

Nature of deletions formed in response to IS2 in a revertant of the gal3 insertion of E. coli.

Summary The gal3 mutation of E. coli, which arose by the insertion of IS2 in the OP region of the gal operon, reverts spontaneously by excision of the IS2 to produce inducible revertants or by mutational alterations of IS2 to produce constitutive revertants. However, gal3() strains bearing chlD-pgl deletions produce constitutive revertants alone. We proposed that deletions formed in the presence of IS2 terminate specifically at its right end, so that revertants arising by excision of IS2 fuse the gal genes to other promoters. Therefore, the revertants are exclusively constitutive.The above hypothesis was tested by electron microscopy of IS2-specific deletions. Spontaneous chlD-pgl deletions were isolated from gal ^c331 (a revertant of gal3 which retains IS2) and transferred to gal genomes. Electron microscopy of DNA heteroduplexes from these phages confirmed that all of the deletions examined have one end-point fixed at the right end of IS2, whereas their other end-points are variable. In each case, the complete IS2 element was apparently retained. This specificity was also detectable in a revertant (gal ^c200) which retains only the right 1/5 portion of the IS2. The frequencies of these deletions were generally increased in constitutive revertants of gal3. Since a galO ^cmutant did not show a similar increase, it seems that this effect depends upon a base sequence provided by IS2. Moreover, the presence of prophage contributes to the specificity and, in some instances, the frequency of IS2-specific deletions.A mechanism for the formation of the IS2-specific deletions has been proposed. A base sequence located at, or near, the right end of IS2 is recognized and nicked by a specific endonuclease. The nick is enlarged by unidirectional, exonucleolytic degradation to produce deletions extending outwards from the insertion. In constitutive revertants, the nicking site may be exposed to endonucleolytic attack more frequently.     

Genetic analysis of a temperature-sensitive Salmonella typhimurium rho mutant with an altered rho-associated polycytidylate-dependent adenosine triphosphatase activity.

A conditional-lethal rho mutant of Salmonella typhimurium LT2 has been isolated. The mutation was selected as a suppressor of the polarity of an insertion sequence (IS)2-induced mutation (gal3) carried on an F' plasmid. In addition to suppression of IS2-induced polarity, the rho-111 mutation suppressed nonsense and frameshift polarity. The rho-associated polycytidylic acid-dependent adenosine triphosphatase activity in the mutant strain was elevated 15-fold above that in the parental strain, and the mutant rho protein was thermally unstable. A temperature-resistant revertant of the mutant strain did not suppress polarity and contained normal levels of polycytidylic acid-dependent adenosine triphosphatase, suggesting that the phenotype of the rho-111-bearing strain is the consequence of a single mutation. The rho-111 mutation was located on the S. typhimurium linkage map midway between the ilv and cya loci by phage P22 cotransduction studies. F' plasmid maintenance was not impaired in the mutant strain, and the mutation was recessive to the wild-type allele. The rho-111 mutation did not alter in vivo expression of either the tryptophan or histidine operons.     

Extraction of β-galactosidase fused protein a in aqueous two-phase systems

A method for the purification of proteins hybridized with β-galactosidase and produced in Escherichia coli is suggested. The method is based on the dominating properties of the β-galactosidase part of the molecule that are utilized for extraction in a poly(ethylene glycol) 4000/potassium phosphate aqueous two-phase system. The purification of the hybrid protein Staphylococcal protein A-Escherichia coli-β-galactosidase (SpA-βgal) produced in Escherichia coli is described. The partitioning of the cell debris and SpA-βgal depended on the distance to the critical point, i.e., the length of the tie line. A poly(ethylene glycol) top phase and an interface free from cell debris were obtained for a composition close to the binodial with a relatively short tie line. At this composition no Spa-βgal partitioned to the interface. When the length of the tie line was increased, more of the SpA-βgal was caught by the interface. The partitioning of SpA-βgal to the top phase was also affected by the salts present during the extraction. The utilization of SpA-βgal for affinity extraction has been investigated. Experiments with SpA-βgal and fluorescence-labeled human IgG(hIgG-F) in a poly(ethylene glycol) 4000/potassium phosphate aqueous two-phase system showed that the complex SpA-βgal-hlgG-F was partitioned to the interface, probably as a precipitate.     

Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.     

Biosynthesis of the major brain gangliosides GD1a and GT1b

Gangliosides-sialylated glycosphingolipids-are the major glycoconjugates of nerve cells. The same four structures-GM1, GD1a, GD1b and GT1b-comprise the great majority of gangliosides in mammalian brains. They share a common tetrasaccharide core (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1'Cer) with one or two sialic acids on the internal galactose and zero (GM1 and GD1b) or one (GD1a and GT1b) α2-3-linked sialic acid on the terminal galactose. Whereas the genes responsible for the sialylation of the internal galactose are known, those responsible for terminal sialylation have not been established in vivo. We report that St3gal2 and St3gal3 are responsible for nearly all the terminal sialylation of brain gangliosides in the mouse. When brain ganglioside expression was analyzed in adult St3gal1-, St3gal2-, St3gal3- and St3gal4-null mice, only St3gal2-null mice differed significantly from wild type, expressing half the normal amount of GD1a and GT1b. St3gal1/2-double-null mice were no different than St3gal2-single-null mice; however, St3gal2/3-double-null mice were >95% depleted in gangliosides GD1a and GT1b. Total ganglioside expression (lipid-bound sialic acid) in the brains of St3gal2/3-double-null mice was equivalent to that in wild-type mice, whereas total protein sialylation was reduced by half. St3gal2/3-double-null mice were small, weak and short lived. They were half the weight of wild-type mice at weaning and displayed early hindlimb dysreflexia. We conclude that the St3gal2 and St3gal3 gene products (ST3Gal-II and ST3Gal-III sialyltransferases) are largely responsible for ganglioside terminal α2-3 sialylation in the brain, synthesizing the major brain gangliosides GD1a and GT1b.     

Analysis of the Gal3 Signal Transduction Pathway Activating Gal4 Protein-Dependent Transcription in Saccharomyces Cerevisiae

The Saccharomyces cerevisiae GAL/MEL regulon genes are normally induced within minutes of galactose addition, but gal3 mutants exhibit a 3-5-day induction lag. We have discovered that this long-term adaptation (LTA) phenotype conferred by gal3 is complemented by multiple copies of the GAL1 gene. Based on this result and the striking similarity between the GAL3 and GAL1 protein sequences we attempted to detect galactokinase activity that might be associated with the GAL3 protein. By both in vivo and in vitro tests the GAL3 gene product does not appear to catalyze a galactokinase-like reaction. In complementary experiments, Escherichia coli galactokinase expressed in yeast was shown to complement the gal1 but not the gal3 mutation. Thus, the complementation activity provided by GAL1 is not likely due to galactokinase activity, but rather due to a distinct GAL3-like activity. Overall, the results indicate that GAL1 encodes a bifunctional protein. In related experiments we tested for function of the LTA induction pathway in gal3 cells deficient for other gene functions. It has been known for some time that gal3gal1, gal3gal7, gal3gal10, and gal3 rho- are incapable of induction. We constructed isogenic haploid strains bearing the gal3 mutation in combination with either gal15 or pgi1 mutations: the gal15 and pgi1 blocks are not specific for the galactose pathway in contrast to the gal1, gal7 and gal10 blocks. The gal3gal5 and gal3pgi1 double mutants were not inducible, whereas both the gal5 and pgi1 single mutants were inducible. We conclude that, in addition to the GAL3-like activity of GAL1, functions beyond the galactose-specific GAL1, GAL7 and GAL10 enzymes are required for the LTA induction pathway.     

Regulation of open complex formation at the Escherichia coli galactose operon promoters. Simultaneous interaction of RNA polymerase, gal repressor and CAP/cAMP.

The regulation of open complex formation at the Escherichia coli galactose operon promoters by galactose repressor and catabolite activator protein/cyclic AMP (CAP/cAMP) was investigated in DNA-binding and kinetic experiments performed in vitro. We found that gal repressor and CAP/cAMP bind to the gal regulatory region independently, resulting in simultaneous occupancy of the two gal operators and the CAP/cAMP binding site. Both CAP/cAMP and gal repressor altered the partitioning of RNA polymerase between the two overlapping gal promoters. Open complexes formed in the absence of added regulatory proteins were partitioned between gal P1 and P2 with occupancies of 25% and 75%, respectively. CAP/cAMP caused open complexes to be formed nearly exclusively at P1 (98% occupancy). gal repressor caused a co-ordinated, but incomplete, switch in promoter partitioning from P1 to P2 in both the absence and presence of CAP/cAMP. We measured the kinetic constants governing open complex formation and decay at the gal promoters in the absence and presence of gal repressor and CAP/cAMP. CAP/cAMP had the largest effect on the kinetics of open complex formation, resulting in a 30-fold increase in the apparent binding constant. We conclude that the regulation of open complex formation at the gal promoters does not result from competition between gal repressor, CAP/cAMP and RNA polymerase for binding at the gal operon regulatory region, but instead results from the interactions of the three proteins during the formation of a nucleoprotein complex on the gal DNA fragment. Finally, we present a kinetic model for the regulation of open complex formation at the gal operon.