Heinz Saedler

IS-Elements in Microorganisms

The bacterial chromosome, while evolving by point mutations in its DNA, is thought to be rather stable with regard to its gross organization. For example, even though the DNAs of Salmonella and Escherichia cross-hybridize only weakly due to divergent sequence evolution (Demerec and New, 1965; Brenner et al., 1969), the genetic maps of the two bacteria are rather similar with respect to the order of functions (Sanderson and Demerec, 1965).

Tn951: A new transposon carrying a lactose operon

SummaryA new transposon, Tn951, is described, which derives from plasmid pGC1, originally isolated from Yersinia enterocolitica. Tn951 is 16.6 kb long and presumably flanked by small inverted repeats. It carries the lac genes i, z and y. This lac system is homologous to the E. coli lac operon. However, homology is restricted to 5.6 kb. The DNA sequences surrounding the lac operons on Tn951 and E. coli are nonhomologous. This leads to speculations about the origin of the E. coli lac operon itself.

Deletions and an inversion induced by a resident IS1 of the lactose transposon Tn951

SummaryDNA-DNA filter binding tests, “Southern” blotting experiments and DNA heteroduplex analysis clearly show that Tn951 contains an IS1 element. This IS1-951 sequence is peculiar in that it does not contain the PstI cleavage site which is usually observed on E. coli derived IS1 elements. Nonetheless, IS1-951 induces deletions. This process is temperature dependent. One instance of an IS1-951 induced inversion was observed, the structure of which is compatible with the current models of transposition of IS elements.

Isolation of the mini insertions IS6 and IS7 of E. coli

SummaryThe mini IS elements IS6 and IS7 have been detected in constitutive gal+ revertants of galOP-308::IS2 (I), in which the expression of the gal operon is turned off by IS2 in orientation I. Both, IS6 and IS7, are integrated into IS2 proximal to the gal structural genes. IS6 is 115 base pairs long and causes 50% constitutive expression of the gal genes. IS7 is only 65 base pairs long and the gal operon is expressed 20% constitutively compared to the gal+ wild type operon. Both IS6 and IS7 are excised frequently, in the absence of selective pressure. These findings are discussed with respect to the evolution of gene expression.

IS2-61 and IS2-611 arise by illegitimate recombination from IS2-6.

SummaryA more stable derivative of IS2-6 has been isolated, which had lost 54 bp of the 108 bp long insert characteristic of IS2-6. This new allele of IS2, IS2-61, segregates the remaining 54 bp to yield allele IS2-611. DNA sequence analysis shows that the segregation products of IS2-6 arise by recA-independent, illegitimate recombination at 9 bp long direct sequence repetitions.

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

SummaryIS2-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.

Multiple integration sites for the lactose transposon Tn951 on plasmid RP1 and establishment of a coordinate system for Tn951

SummaryVarious molecules generated by transposition of the lactose transposon Tn951 from plasmid pGC1 to plasmid RP1 were examined by DNA heteroduplex and restriction endonuclease analysis. Tn951 was found to transpose to at least eight different sites on RP1 in both possible orientations. A coordinate system for the lactose transposon Tn951 is constructed.

Mapping and characterization of an E. coli mutant defective in IS1-mediated deletion formation

SummaryA mutant of E. coli has been isolated in which the frequency of IS1-mediated deletion formation is reduced as much as 100 fold. The mutation causing this reduction, designated del, was mapped to a position near 61 min, in the vicinity of lysA and galR. Strains carrying a deletion of the del gene were constructed, and these exhibit a significant reduction in the frequency of IS1 excision in addition to impairment of deletion formation. A λ bacteriophage capable of transducing lysA and del was also isolated.

Homeotic Genes in the Genetic Control of Flower Morphogenesis in Antirrhinum majus

“Anyone who pays a little attention to the growth of plants will readily observe that certain of their external members are sometimes transformed, so that they assume — either wholly or in some lesser degree — the form of the members nearest the series”.

Properties of DNA Insertion Elements of E. coli

The presence of IS elements in the chromosome of E.coli was originally revealed by the transposition of these DNA elements from their natural positions into indicator systems, resulting in a recognizable mutant phenotype. If, for example, the gal operon is used as an indicator system, mutations can be isolated in which not only one of the three structural genes of the galactose operon is inactivated, but the expression of the Promoter distal genes is also abolished. These mutations therefore are strongly polar. The analysis of the nature of such mutations is facilitated by the existence of the gal transducing phage λ and the development of techniques for examining heteroduplex DNA in the electron microscope. With the help of these tools it is possible to inspect hybrid DNA molecules consisting of one DNA strand carrying the strongly polar mutation and a complementary strand of λ dgal in the electron microscope. The strongly polar mutation is seen as a single stranded DNA loop emerging from a position of the double stranded heteroduplex molecule, which corresponds to the map position of the mutation. Analysing various independently isolated strongly polar mutations with the above technique, revealed the existence of different categories of IS elements.