Molecular cloning of cDNAs for auxin-induced mRNAs and developmental expression of the auxin-inducible genes

By differential hybridization, two auxin-inducible cDNA clones (SAR1 and SAR2) have been isolated from a cDNA library constructed to poly(A)⁺ mRNA from auxin-treated strawberry receptacles. Both the clones have been used as probes to study the expression of the auxin-induced genes in pollinated and unpollinated fruits of various stages of development and in different organs. A high level of auxin-induced mRNAs is found in pollinated fruits as compared to unpollinated fruits of the same age, suggesting that the expression of the auxin-induced genes is developmentally regulated and the level of auxin-induced mRNAs is regulated by endogenous auxin. Furthermore, our data on the expression of SAR1 and SAR2 genes in pollinated and unpollinated fruits revealed a positive correlation between growth of strawberry fruit and the induction of mRNA corresponding to the SAR1 and SAR2 clones. Ethylene has no effect on the expression of the auxin-induced mRNAs. SAR1 mRNA is not detected in other parts of strawberry plants whereas SAR2 mRNA is present in roots. Furthermore, mRNA corresponding to SAR1 and SAR2 is not detected in other auxin-responsive plant systems such as pea epicotyls and bean explants.     

Evidence that pretreatment of Escherichia coli cells with N-methyl-N''-nitro-N-nitrosoguanidine enhances mutability of subsequently infecting phage lambda

Summary The frequency of mutations in is significantly higher for host cells of a rec ⁺ or recA ^- strain pretreated with N-methyl-N-nitro-N-nitrosoguanidine (NG) than for untreated control cells. Direct NG treatment of DNA-injected host cells results in about tenfold higher mutation frequency in than NG treatment of host cells alone. Since mutability of is enhanced by UV preirradiation of host cells in the rec ⁺ strain but not in the recA ^- strain, indirect NG mutagenesis is different in mechanism from indirect UV mutagenesis. It is concluded that NG mutagenesis in consists of at least two processes: induction of rec ⁺-independent mutagenic activity in the host bacterium and production of rec ⁺-independent mutational damage to intracellular DNA.     

Rearrangements between the operators in the bacteriophage lambda

Summary The left operator mutant v2s develops poorly during infection as a result of constitutive expression of the left operon. A revertant of v2s, designated iri, was found to contain an inversion of the cI region with the inversion endpoints to be within the lambda operators o _L and o _R. Formation of the inversion is facilitated by a translocation of right operator o _R ^c mutant sequence to the left operator in v2s. The inversion in iri positions wild-type o _R sequence at o _L returning control of the left operon to repression by the lambda cro repressor.     

A hotspot of spontaneous and UV-induced illegitimate recombination during formation ofλbio transducing phage

To study the mechanism of spontaneous and UV-induced illegitimate recombination, we examined the formation of thebio specialized transducing phage inEscherichia coli. Because mostbio transducing phages have double defects in thered andgam genes and have the capacity to form a plaque on anE. coli P2 lysogen (Spi^– phenotype), we selectedbio transducing phage by their Spi^– phenotype, rather than using thebio marker. We determined sequences of recombination junctions ofbio transducing phages isolated with or without UV irradiation and deduced sequences of parental recombination sites. The recombination sites were widely distributed onE. coli bio and DNAs, except for a hotspot which accounts for 57% of UV-inducedbio transducing phages and 77% of spontaneously inducedbio transducing phages. The hotspot sites onE. coli and DNAs shared a short homology of 9 bp. In addition, we detected direct repeat sequences of 8 by within and near both thebio and hotspots. ArecA mutation did not affect the frequency of the recombination at the hotspot, indicating that this recombination is not a variant ofrecA-dependent homologous recombination. We discuss a model in which the short homology as well as the direct repeats play essential roles in illegitimate recombination at the hotspot.     

Physical characterisation of the "Rac prophage" in E. coli K12.

Summary We confirm the hypothesis of Low (1973) that many E. coli K 12 strains contain a prophage (the Rac prophage) located a few minutes clockwise of the trp operon on the genetic map. We have used restriction endonucleases and ³²P-labelled probes to construct a physical map of this prophage. Some E. coli K 12 strains, including AB1157, have lost the entire prophage, apparently by a specific deletion. This is consistent with prophage excision by site-specific recombination. reverse (rev) phages (Zissler et al., 1971) are recombination proficient derivatives of phage in which the phage recombination functions have been replaced by analogous functions (RecE) derived from the host chromosome (Gottesman et al., 1974; Gillen et al., 1977). Our data support the origin of rev phages by recombination between and the Rac prophage following excision of the Rac prophage from the E. coli chromosome.Important experimental data are included in the Figure legends.     

Structure and function of the repressor of bacteriophage lambda

Summary The high-affinity mutant cI gene of cIha (Nag et al. 1984) was cloned in the multicopy plasmid pBR322. In the resulting plasmid, pMD 102, a lacUV5 promoter was inserted giving the lacUV5-cIha fusion plasmid pMD 205. Bacteria carrying pMD 102 and pMD 205 contain 2.5 and 15 times, respectively, the level of repressor in a monolysogen of cIha. Results of the study of certain properties of the bacteria carrying these plasmids suggest that the ha repressor also has a higher affinity for the virulent mutant operators as well as the prm promoter of .     

The glutamine synthetase gene family of Arabidopsis thaliana: light-regulation and differential expression in leaves, roots and seeds.

Summary Glutamine synthetase (GS) plays an important role in the assimilation of nitrogen by higher plants. We present here a molecular analysis of the GS polypeptides, mRNAs, and genes of Arabidopsis thaliana. Western blot analysis of leaf and root protein extracts revealed at least two distinct GS polypeptides; 43 kDa and 39 kDa GS polypeptides were present in leaves, while only a 39 kDa GS was detected in roots. The 43 kDa GS polypeptide is light-inducible. In etiolated seedlings only the 39 kDa GS was detected. However, upon greening the 43 kDa GS increased to levels comparable to those observed in light-grown plants. Four distinct GS cDNA clones, Atgsl1, Atgsrl, Atgsr2 and Atk6 were isolated and characterized. Their complete nucleotide and deduced amino acid sequences are presented. The coding sequences of the four clones are 70–88% similar while their 5 and 3 untranslated regions exhibit less than 50% similarity. Northern blots of leaf, root and germinated seed RNA revealed that the four cDNAs hybridize to mRNAs which are differentially expressed in the organs of Arabidopsis thaliana. Atgsl1 is leaf-specific and hybridizes to a 1.6 kb mRNA. Both Atgsr1 and Atgskb6 hybridize to 1.4 kb mRNAs which are expressed in both roots and germinated seeds. Atgsr2 hybridizes to a 1.4 kb mRNA, which is primarily expressed in roots with low levels of expression in seeds and leaves. Atgsl1, which represents the leaf-specific mRNA, is induced by light. Atgsl1 mRNA levels increase during the greening of etiolated seedlings while Atgsr1 levels remain constant. Southern blot analysis indicated that the Arabidopsis genome contains at least four and possibly five distinct GS genes.     

A transducing lambda phage carrying grpE, a bacterial gene necessary for lambda DNA replication, and two ribosomal protein genes, rpsP (S16) and rplS (L19).

Summary A grpE mutation of Escherichia coli K12, which blocks DNA replication of the phage (Saito and Uchida, 1977), was mapped at 56 min on the standard genetic map. A transducing phage, grpE22, carrying the wild type allele of the grpE gene was constructed in vitro. Structures of grpE22 and its viable deletion derivatives were determined by electron microscopic analyses of appropriate heteroduplexes. Proteins coded by the bacterial DNA incorporated into the transducing phages were detected by two-dimensional gel electrophoresis. The results showed that the product of the grpE gene is a weakly acidic protein of molecular weight 24,000. Structural genes for two ribosomal proteins, rplS (L19) and rpsP (S16) were also shown to be carried by grpE22.     

Prophage inactivation in recB-proficient Escherichia coli K12 (lambda) lysogens after ultraviolet irradiation

Summary If ultraviolet irradiated, -lysogenic Escherichia coli K12 bacteria are incubated for 4 to 6 h at 30° C, prophage becomes inactive in the non-surviving cells. This was demonstrated by the use of cIts857ind1 prophage which can be induced by heat but not by ultraviolet light. An analysis with various bacterial mutants showed that RecBC recombination enzyme is required in conjunction with RecA protein for prophage inactivation.     

Negative complementation of recA protein by recA1 polypeptide: in vivo recombination requires a multimeric form of recA protein

Summary Recombination in vivo was studied in recA ^- heterozygous lacZ merodiploids by performing -galactosidase assays after infection with precA ⁺. Recombination as measured by -galactosidase production was a linear function of pecA ⁺ multiplicity of infection (MOI) when the strain contained a deletion of the chromosomal recA gene. However, when the strain carried a recA1 missense allele, a higher precA ⁺ MOI was required to obtain levels of recombination comparable to the (recA) strain, and the slope of the dose-response curve increased to approximately two. It is proposed that negative complementation occurs in mixed tetramers of wild-type and missense recA polypeptides, and that in vivo recombination is a property of a multimeric form of recA protein.     

Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis

Summary A 24 kilobase pair region of the E. coli chromosome surrounding the dnaG gene has been cloned and characterized. A phage library was first constructed by ligating a Sau3A (GATC) partial DNA digest of the entire E. coli chromosome into the BamHI (G GATCC) cloning vector charon 28. Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome. This library was probed with a nick-translated plasmid (pRRB1) containing the rpoD gene, which maps adjacent to dnaG at 66 min. Four bacteriophages: 3, 4, 5, 6 that hybridized to the probe were isolated from the 2,500 plaques screened. One phage recombinant 4, was shown to contain the dnaG gene. Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of 4 using different restriction fragments. Plasmids pGL444 and pBS105 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated. The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement a dnaGts chromosomal marker at nonpermissive 40° C. In this manner a correlated physical and genetic map of dnaG was determined. A large number of Tn5 inserts map to a specific 900 b.p. region which we propose may be involved in the regulation of dnaG gene expression.     

The photic environment and scotopic visual pigments of the creek chub,Semotilus atromaculatus and white sucker,Catostomus commersoni

Summary Scotopic visual pigments measured in the creek chub and the white sucker are porphyropsins with mean _max values located at 538.3 and 536.5 nm, respectively. There is a shift of the _max towards shorter wavelengths during the winter in both of these species coinciding with similar changes in the quality of downwelling light. _max is significantly correlated to the P₅₀ and spectrum width of the downwelling light and dissolved oxygen. An analysis of variance shows that there are significant differences between the _max values of: fish in the two lakes, fish at different times, the two species at different times and fish in different lakes at different seasons. The offset visual pigments of both species appear to be well adapted to their photic environment in terms of the contrast hypothesis. This improvement of contrast detection is discussed in relation to their feeding habits.Abbreviations _max wavelength at which absorbance is maximum - P₅₀ wavelength which halves the total number of photons between 400 and 700 nm, a measure of spectral quality - PAR photosynthetically active radiation - MSP microspectrophotometric - SE standard error     

Cloning of the replication gene P of bacteriophage lambda: Effects of increased P-protein synthesis on cellular and phage DNA replication

Summary A restriction fragment of DNA carrying the P gene was cloned in the high copy number plasmid RSF2124. Cells harbouring this new plasmid RSF2124/E complement Pam80 phage. A lac promoter-operator region (lacP), produced by EcoRI digestion of plasmid pKB252, was inserted into RSF2124/glE such that induction of the lac promoter by IPTG or lactose leads to increased production of the P gene product. A high amount of P protein in E. coli cells results in a slow inhibition of bacterial DNA synthesis, suggesting that the initiation reaction is blocked by P protein. Synthesis of DNA proceeds normally under these conditions.Nonsuppressing groPA15 mutant bacteria which are unable to support the replication of wild-type (wt), acquire the ability to replicate Pam80 phage but not wt when they are transformed with a plasmid carrying the P gene. When harbouring a plasmid containing the mutant Pamber 80 gene, groPA15 mutants are able to support the replication of wt phage when infected at a high multiplicity. Pam80 phage does not multiply in these cells.     

Fluorescence behavior of tryptophan residues of bovine and human serum albumins in ionic surfactant solutions: A comparative study of the two and one tryptophan(s) of bovine and human albumins

The fluorescence behavior of two tryptophans (Trp-134, Trp-213) in bovine serum albumin (BSA) and a single tryptophan (Trp-214) in human serum albumin (HSA) was examined. The maximum emission wavelength (_max) was 340.0 nm for both proteins. In a solution of sodium dodecyl sulfate (SDS), the _max of BSA abruptly shifted to 332 nm at 1 mM SDS and then reversed to 334 nm at 3 mM SDS. The _max of HSA gradually shifted to 330 nm below 3 mM SDS, although it returned to 338 nm at 10 mM SDS. In contrast to this, in a solution of dodecyltrimethylammonium bromide, the _max positions of BSA and HSA gradually shifted to 334.0 and 331.5 nm, respectively. Differences in the fluorescence behavior of the proteins are attributed to the fact that Trp-134 exists only in BSA, with the assumption that Trp-213 of BSA behaves the same as Trp-214 of HSA. The Trp-134 behavior appears to relate to the disruption of the helical structure in the SDS solution.     

Inactivation of DNA-binding activity of repressor in extracts of lambda-lysogen treated with mitomycin C

Summary Crude extracts from -lysogens treated with mitomycin C were prepared, and immunity repressor levels in the extracts were assayed by the binding activity specific to DNA immunity region. It has been shown that while the repressor levels in the extracts from C600(+) are reduced after mitomycin C treatment, the levels in C600recA(+) and C600 C72(+) which have defects in lifting the immunity are not affected by the treatment. The repressor levels in the extracts prepared from C600 T44(+) after temperature shift up, whose prophage is inducible at high temperature, are also reduced. From the study of chloramphenicol effect, it was indicated that de novo protein synthesis is required for the inactivation of the repressor in C600(+) by mitomycin C, but not in T44(+) by high temperature.     

An improved selection method for λlacZ − phages based on galactose sensitivity

The determination of thelacZ mutant frequency in gt10lacZ phage vectors isolated from the transgenic mouse strain 40.6 (MutaMouse), requires the screening of large numbers of phages on -galactosidase activity. Existing methods rely on distinguishing a few white plaques on X-gal containing plates amongst a multide of blue ones which is both time-consuming and expensive. The new screening method described here employs the galactose sensitiveEscherichia coli C lacZ recA galE strain into which a multicopy plasmid has been introduced, which results in over-expression of thegalK andgalT genes. In the presence of phenyl--d-galactopyranoside, a substrate for -galactosidase, this leads to the suppression of lacZ ⁺ phage propagation without affecting the ability of lacZ ^– phages to form plaques. With this method it is possible to screen 1.5×10⁶ phages on a single 9-cm Petri dish. Furthermore, the need for blue/white screening has been eliminated.