Tn10 insertions in the pfkB region of Escherichia coli.

The locus pfkB is known to determine expression of a minor phosphofructokinase (Pfk-2). Pfk-2 and pfkB seem to be dispensable, since Tn10 insertions in pfkB, as well as deletions from Tn10 nearby, are obtainable. Strains deleted for both pfkA and pgkB are unable to grow at all on sugars whose primary route of metabolism is via fructose 6-phosphate, confirming earlier reports implicating the low Pfk-2 activity, rather than the pentose-phosphate pathway, as needed for the slow growth on sugars of pfkA pfkB+ strains. The pfkB locus probably contains the structural gene for Pfk-2, since a mutation closely linked to pfkB1, which affects growth on glycerol, is found to alter the enzyme. Partial phenotypic suppression of the pfkA mutant phenotype results from Tn10 insertion very close to the pps gene, ca. 0.5 min from pgkB. The insertion does not clearly affect either Pfk-2 or phosphoenolpyruvate synthetase, and the mechanism of suppression is unclear.     

Nucleotide sequence of gene pfkB encoding the minor phosphofructokinase of Escherichia coli K-12

The nucleotide sequence of a 1.3-kb DNA fragment containing the entire pfkB gene which codes for Pfk-2 of Escherichia coli, a minor phosphofructokinase (Pfk) enzyme, is reported. The Pfk-2 protein subunit is encoded by 924 bp, has 308 amino acids and an Mr of 33 000. Like other weakly expressed E. coli genes the codon usage in the pfkB gene is random; there is no strong bias for the usage of major tRNA isoaccepting species, and the codon preference rules of Grosjean and Fiers [Gene, 18 (1982) 199-209] are followed. This is the first report of the complete gene sequence of a phosphofructokinase.     

Interaction between the replication origin and the initiator protein of the filamentous phage f1. Binding occurs in two steps

The replication initiator protein of bacteriophage f1 (gene II protein) binds to the phage origin and forms two complexes that are separable by polyacrylamide gel electrophoresis. Complex I is formed at low gene II protein concentrations, and shows protection from DNase I of about 25 base-pairs (from position +2 to +28 relative to the nicking site) at the center of the minimal origin sequence. Complex II is produced at higher concentrations of the protein, and has about 40 base-pairs (from -7 to +33) protected. On the basis of gel mobility, complex II appears to contain twice the amount of gene II protein as does complex I. The 40 base-pair sequence protected in complex II corresponds to the minimal origin sequence as determined by in-vivo analyses. The central 15 base-pair sequence (from +6 to +20) of the minimal origin consists of two repeats in inverted orientation. This sequence, when cloned into a plasmid, can form complex I, but not complex II. We call this 15 base-pair element the core binding sequence for gene II protein. Methylation interference with the formation of complex I by the wild-type origin indicates that gene II protein contacts six guanine residues located in a symmetric configuration within the core binding sequence. Formation of complex II requires, in addition to the core binding sequence, the adjacent ten base-pair sequence on the right containing a third homologous repeat. A methylation interference experiment performed on complex II indicates that gene II protein interacts homologously with the three repeats. In complex II, gene II protein protects from DNase I digestion not only ten base-pairs on the right but also ten base-pairs on the left of the sequence that is protected in complex I. Footprint analyses of various deletion mutants indicate that the left-most ten base-pairs are protected regardless of their sequence. The site of nicking by gene II protein is located within this region. A model is presented for the binding reaction involving both protein-DNA and protein-protein interactions.     

Recognition and cleavage of the bacteriophage P1 packaging site (pac). II. Functional limits of pac and location of pac cleavage termini

Bacteriophage P1 initiates the processive packaging of its DNA at a unique site called pac. We show that a functional pac site is contained within a 161 base-pair segment of P1 EcoRI fragment 20. It extends from a position 71 base-pairs to a position 232 base-pairs from the EcoRI-22 proximal side of that fragment. The 3' and 5' pac termini are located centrally within that 161 base-pair region and are distributed over about a turn of the DNA helix. The DNA sequence of the terminus region is shown below, with the large arrows indicating the positions of termini that are frequently represented in the PI population and the small arrows indicating the positions of termini that are rarely represented in the P1 population. (Sequence: in text). Digestion of P1 virus DNA with EcoRI generates two major EcoRI-pac fragments, which differ in size by about five or six base-pairs. While the structure and position of the double-stranded pac ends of these fragments have not been determined precisely, the 5' termini at those ends probably correspond to the two major pac cleavage sites in the upper strand of the sequences shown above. The 161 base-pair pac site contains the hexanucleotide sequence 5'-TGATCAG-3' repeated four times at one end and three times at the other. Removal of just one of those elements from either the right or left ends of pac reduces pac cleavage by about tenfold. Moreover, the elements appear to be additive in their effect on pac cleavage, as removal of one and a half elements or all three elements from the right side of pac reduces pac cleavage 100-fold, and greater than 1000-fold, respectively.     

Cloning and expression of Clostridium pasteurianum galactokinase gene in Escherichia coli K-12 and nucleotide sequence analysis of a region affecting the amount of the enzyme

The Clostridium pasteurianum galactokinase gene was cloned by complementation, of the galK locus, into Escherichia coli. Restriction enzyme analysis subcloning and Tn5 mutagenesis indicated that the gene was located on a 1.8 X 10(3) base-pair ClaI-Sau3A fragment that encoded a polypeptide of approximately 40 Mr. Although the C. pasteurianum and the E. coli galactokinases have similar subunit molecular weights, Southern hybridization analysis indicated no strong homology between their genes. Even though this clone showed a low level of galactokinase expression, the Gal+ phenotype, provided by the clostridial galactokinase, was unstable in E. coli, and the gene was frequently inactivated by the spontaneous acquisition of insertion sequences. A second clone containing this gene on a large restriction fragment was isolated by hybridization. This clone was unable to grow on galactose-containing media due to the overproduction of galactokinase. Comparison of the plasmids from these two clones revealed that the second contained an additional 300 base-pairs located at one end of the galactokinase gene. Appropriate operon fusions with a promoter-less E. coli galactokinase gene indicated that these additional 300 base-pairs had promoter activity in E. coli. The DNA sequence of this region which lies upstream of the C. pasteurianum galactokinase gene was determined and compared with that from several clones producing high, low or undetectable amounts of galactokinase. The reasons for the high and low level expression and for the instability of the C. pasteurianum galactokinase in E. coli are discussed. The presence of the galactokinase suggests that galactose is used in C. pasteurianum through the Leloir pathway via galactose 1-phosphate.     

Evolution of the primate beta-globin gene region. High rate of variation in CpG dinucleotides and in short repeated sequences between man and chimpanzee

A 5500 base-pair fragment including the beta-globin gene downstream from codon 122 and about 4000 base-pairs of its 5' flanking sequence was cloned from chimpanzee DNA and thoroughly sequenced before being compared with the corresponding human sequence: 88 point differences (83 substitutions and 5 deletions or insertions of 1 base-pair) were detected as well as seven more important deletion/insertion events. These changes occur preferentially in two kinds of structure. First, 40% of the CpG dinucleotides present in either human or chimpanzee sequences are affected by nucleotide variations. This corresponds to a divergence level considerably higher than that expected. Second, most short repeated sequences found in the 5' extragenic sequence are involved in mutational events (amplification or contraction of the number of basic motifs as well as point substitutions or deletions/insertions of 1 base-pair). Considering the very low level of nucleotide sequence divergence between these two closely related species, our data provide direct evidence for CpG and tandem array instability.     

Phosphofructokinases from Escherichia coli. Purification and characterization of the nonallosteric isozyme.

The main phosphofructokinase of Escherichia coli (PFK I) is an extensively studied allosteric enzyme specified by the pfkA gene. A nonallosteric phosphofructokinase was reported (Fraenkel, D.G., Kotlarz, D., and Bluc, H. (1973) J. Biol. Chem. 248, 4865-4866) in strains carrying the pfkB1 mutation, a suppressor of pfkA mutants, and very low levels of this enzyme have also been detected in strains not carrying the suppressor (i.e. pfkB+). The nonallosteric protein has now been prepared pure from three strains, one carrying pfkB1 and pfkA+, one carrying pfkB1 and completely deleted for pfkA, and one carrying pfkB+ and also deleted for pfkA. It is apparently the same enzyme (PFK II) in all three strains, which shows that pfkB1 is a mutation affecting the amount of a normally minor isozyme. PFK II is a tetramer of slightly larger subunit molecular weight than PFK I (36,000 and 34,000, respectively). No immunological cross-reactivity was detected between PFK II and PFK I. Unlike PFK I, PFK II does not show cooperative interactions with fructose-6-P, inhibition by P-enolpyruvate, or activation by ADP. Also unlike PFK I, PFK II is somewhat sensitive to inhibition by fructose-1,6-P2 and can use tagatose-6-P as substrate. Both enzymes can perform the reverse reaction, fructose-6-P + ATP from fructose-1,6-P2 + ADP in vitro, but not in vivo. The normal function of PFK II is not known.     

The genetic behaviour of a cloned mouse ribosomal DNA segment mimics mouse ribosomal gene evolution.

A 1.7 × 10³ base-pair SalI fragment of mouse ribosomal gene spacer undergoes recA-independent deletions of DNA in units of approximately 126 base-pairs when cloned in λ or bacterial plasmids. When we examined the structure of the 1.7 × 10³ base-pair piece with PvuII we found it to be composed of about equal numbers of copies of each of two subrepeating units, 120 and 130 base-pairs in size. The correlation between the size of the structural subunits and the functional genetic unit of this fragment as expressed in Escherichia coli led us to study the organization of these sequences in mice. SalI (or HindII) digests of DNA samples from wild and inbred strains revealed extensive heterogeneity in the size of fragments homologous to this 1.7 × 10³ base-pair piece. A total of 15 different size classes were detected in our samples. We found that these fragments were also organized in PvuII repeating units about equal in size to the PvuII repeats in the cloned 1.7 × 10³ base-pair piece. Using an objective analytical procedure (see the Appendix) we determined that the 15 different fragments found in our mouse DNA samples probably originated as a result of genetic events based on a 135 base-pair structural unit.We consider the similarity between the size of the PvuII structural unit and the unit of genetic behavior in both the cloned and uncloned DNA samples to be significant. We suspect that there are aspects of the nucleotide structure or organization of the PvuII repeating units that play a dominant role in its genetic behavior, regardless of whether these sequences are present in E. coli or mice. We believe that the clones containing this mouse sequence may provide an experimental system for studying the nature of the genetic events that are involved in multigene evolution.     

PfkB and pfkC loci of Escherichia coli.

Mutants lacking Escherichia coli phosphofructokinase (pfkA, 78 min) are suppressed by the unlinked pfkB1 mutation, which restores some enzyme activity (Morrissey and Fraenkel, 1972). We here describe a secondary mutation at pfkB, "PFKB-," which abolishes the suppression as well as the low residual activity of unsuppressed pfkA mutants. pfkB is at about 33 min. with the gene order groD-pps-pheS-pfkB. A positive selection was found that yielded both the pfkB-mutations and a new similar mutation, pfkC-. pfkC is an early marker in Hfr HL16(ca. 50 to 55 min). Some pfkC-, but no pfkB-, mutations were amber. A temperature-sensitive pfkB- was also obtained. Strains carrying pfkB- or pfkC-, but wild type at pfkA, were not markedly affected in growth on sugars. A new search for suppressors such as pfkB1 gave five independent candidates, all of which suppressed both pfkA1 and pfkA2 and occurred in the pfkB region; none occurred at pfkC. Neither the pfkB nor the pfkC loci have assigned functions. It is likely that they are somehow involved in expression of phosphofructokinase activity 2 (Fraenkel, Kotlarz, and Buc, 1973).     

The 1723 element: a long, homogeneous, highly repeated DNA unit interspersed in the genome of Xenopus laevis

We describe a highly repeated DNA element in the Xenopus laevis genome. This sequence, named the 1723 element, was first identified among sequences that are transcribed during embryonic development. The element is present in about 8500 copies per haploid genome, which together accounts for about 2.4% of the genome. Most copies of the element have highly conserved restriction maps, and are interspersed in the genome. The copies range in size from 6000 to 10,000 base-pairs due to an expandable region that contains variable numbers of a tandemly repeating 183 to 204 base-pair unit. The element is framed by an imperfect 18 base-pair inverted sequence, and inverted repeats of 180 to 185 base-pairs are nearby. Sequence analysis of DNA adjacent to three cloned elements shows that the elements are flanked by 8 base-pair direct repeats. These and other properties of 1723 suggest that it may be transposable.     

Nucleotide sequence of terminal repeats of 412 transposable elements of Drosophila melanogaster: A similarity to proviral long terminal repeats and its implications for the mechanism of transposition

We have sequenced the long terminal direct repeats (and adjacent DNA) of two members of the 412 family of transposable elements of Drosophila melanogaster cloned on fragments of DNA from strain Oregon R. The repeats of the first element are identical and 481 base-pairs long; the repeats of the second are also identical but are 571 base-pairs long. The first 482 base-pairs of the 571 base-pair sequence correspond to the 481 base-pair repeat differing by five base substitutions and one addition/deletion. The 571 base-pair repeats are rare. Each of these 412 elements is flanked by a four base-pair direct repeat, suggesting that insertion of a 412 element is associated with duplication of four base-pairs. Analysis of the “empty site” from strain Canton S corresponding to one of these elements supports this conclusion. The sequence of 481 base-pair repeats and of 412 DNA immediately adjacent to them show striking similarities to corresponding regions of vertebrate proviruses and we discuss the implications this may have for the mechanism of transposition.     

PfkA locus of Escherichia coli.

pfkA was know, on the basis of three mutants, as the likely locus of phosphofructokinase in Escherichia coli, and the unlinked pfkB1 mutation suppressed these mutations by restoring some enzyme activity (Morrissey and Fraenkel, 1972). We now report a new search for the complete inactivation of pfkA (e.g., by deletion or amber mutation), done to assess whether the pfkB1 suppression is by an independent enzyme, phosphofructokinase activity 2 (Fraenkel, Kotlarz, and Buc, 1973). Ten new phosphofructokinase mutants all were at pfkA, rather than at pfkB or pfkC. One of them (pfkA9) gave temperature-sensitive reverants with heat-labile enzyme. Another (pfkA11) proved genetically to be a nonsense mutation, but showed no restored activity when suppressed by supF. However, even unsuppressed it was found to contain an enzyme related to phosphofructokinase activity 1 kinetically (more allosteric), physically (almot identical subunit), and antigenically. All the pfkA mutants apparently contained cross-reacting material to activity 1. All (including pfkA11) were suppressed by the pfkB1 mutation. Several results support the idea that pfkA is the structural gene for the main phosphofructokinase of E. coli (activity 1), but that there is some restriction to its complete inactivation.     

An intron nucleotide sequence variant in a cloned beta +-thalassaemia globin gene.

A 7.5 kb Hsu I restriction fragment of genomic DNA containing a beta-globin gene has been isolated from a patient doubly heterozygous for beta + thalassaemia and a delta beta (Lepore globin fusion gene. This fragment must be derived from the chromosome carrying the beta +-thalassaemia determinant. The gross structure of the cloned gene plus flanking sequences is indistinguishable from that of a normal beta-globin gene. Within in 1606 base-pair transcribed region of the gene there is only one nucleotide difference from the normal beta-globin gene sequence. This is a G leads to A replacement 21 nucleotides upstream from the 3' terminus of the small intron. This nucleotide lies within a 10 base-pair sequence repeated in an inverted configuration near the 5' terminus of the small intron. The nucleotide replacement may result in a precursor mRNA less amenable to RNA splicing than its normal counterpart.     

Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for Pseudomonas isolated from Pseudomonas syringae patovar savastanoi.

The sequence of a 1823 base-pair region containing the replication functions of pPS10, a narrow host-range plasmid isolated from a strain of Pseudomonas savastanoi, is reported. The origin of replication, oriV, or pPS10 is contained in a 535 base-pair fragment of this sequence that can replicate in the presence of trans-acting function(s) of the plasmid. oriV contains four iterons of 22 base-pairs that are preceded by G+C-rich and A+T-rich regions. A dnaA box located adjacent to the repeats of the origin is dispensable but required for efficient replication of pPS10; A and T are equivalent bases at the 5' end of the box. repA, the gene of a trans-acting replication protein of 26,700 Mr has been identified by genetic and functional analysis. repA is adjacent to the origin of replication and is preceded by the consensus sequences of a typical sigma 70 promoter of Escherichia coli. The RepA protein has been identified, using the minicell system of E. coli, as a polypeptide with an apparent molecular mass of 26,000. A minimal pPS10 replicon has been defined to a continuous 1267 base-pair region of pPS10 that includes the oriV and repA sequences.