Derivation of the relationship between neutral mutation and fixation solely from the definition of selective neutrality.

A mutation whose fixation is independent of natural selection is termed a neutral mutation. Therefore selective neutrality of a mutation can be defined by independence of its fixation from natural selection. By the population genetic approach, Kimura [Kimura, M. (1962) Genetics 47, 713-719] predicted that the probability of fixation of a neutral mutation (u) is equal to the frequency of the new allele at the start (p). The approach traced the temporal sequence of the fixation process, and the prediction was obtained by assuming the selective equality of neutral mutant and wild-type alleles during the fixation process. The prediction, however, has not been verified by observation. In the present study, I search for the mathematical equation that represents the definition of selective neutrality. Because the definition concerns only mutation and fixation, an ideal approach should deal only with these and not with the intervening process of fixation. The approach begins by analysis of the state of fixation of a neutral mutation, and its relation with the initial state is deduced logically from the definition. The approach shows that the equality of the alleles during the fixation process is equivalent to the equality of probability of their ultimate fixation in a steady state. Both are manifestations of the definition of selective neutrality. Then, solely from this dual nature of the definition, the equality between u and p is derived directly. Therefore, the definition of selective neutrality can be represented by the equation u = p.

Complex formation and functional versatility of Mre11 of budding yeast in recombination.

Meiotic recombination of S. cerevisiae contains two temporally coupled processes, formation and processing of double-strand breaks (DSBs). Mre11 forms a complex with Rad50 and Xrs2, acting as the binding core, and participates in DSB processing. Although these proteins are also involved in DSB formation, Mre11 is not necessarily holding them. The C-terminal region of Mre11 is required only for DSB formation and binds to some meiotic proteins. The N-terminal half specifies nuclease activities that are collectively required for DSB processing. Mre11 has a DNA-binding site for DSB formation and another site for DSB processing. It has two regions to bind to Rad50. Mre11 repairs methyl methanesulfonate-induced DSBs by reactions that require the nuclease activities and those that do not.

Structure of the ColE1 DNA molecule before segregation to daughter molecules.

The segregation of daughter DNA molecules at the end stage of replication of plasmid ColE1 was examined. When circular ColE1 DNA replicates in a cell extract at a high KCl concentration (140 mM), a unique class of molecules accumulates. When the molecule is cleaved by a restriction enzyme that cuts the ColE1 DNA at a single site, an X-shaped molecule in which two linear components are held together around the origin of DNA replication is made. For a large fraction of these molecules, the 5' end of the leading strand remains at the origin and the 3' end of the strand is about 30 nucleotides upstream of the origin. The 3' end of the lagging strand is located at the terH site (17 nucleotides upstream of the origin) and the 5' end of the strand is a few hundred nucleotides upstream of the terH site. Thus the parental strands of the molecule intertwine with each other only once. When the KCl concentration is lowered to 70 mM, practically all of these molecules are converted to daughter circular monomers or to catenanes consisting of two singly interlocked circular units.

Functional structures of the recA protein found by chimera analysis.

We developed a novel genetic method for finding functional regions of a protein by the analysis of chimeras formed between homologous proteins. Sets of chimeric genes were made by intramolecular homologous recombination in a linearized plasmid DNA carrying both recA genes of Escherichia coli and Pseudomonas aeruginosa. A recBCsbcA strain of E. coli was used for isolation of plasmids carrying recombinants between these genes. Examination of properties of E. coli strains deleting the recA gene and carrying a plasmid with a chimeric gene shows that chimera formation at certain positions inactivates a RecA function. Frequently, all chimeras with a junction in a certain region of the protein inactivate a function. Rather than a direct effect of the presence of the junction at a particular position, mismatching of the regions both sides of the junction that are derived from the different species is responsible for the inactivation. For a chimeric protein to be functional, certain pairs of sequences in different regions of the protein must derive from the same parent. Four pairs of such sequences were found: two are involved in activities for genetic recombination and for resistance to ultraviolet light irradiation and the others in formation of active oligomers. Regions defined by these sequences are located in the looped regions of the protein. A pair of regions may co-operate to form a functional folded structure.

Complexes formed by complementary RNA stem-loops. Their formations, structures and interaction with ColE1 Rom protein.

Regulation of replication of plasmid ColE1 involves interaction of two plasmid-specified RNA transcripts. One of these RNAs (RNA II) serves as a primer for DNA synthesis, and the other (RNA I) is complementary to part of RNA II. The complementary regions of RNA I and RNA II form several stem-loop structures. Binding of these RNAs that regulates DNA replication begins by interaction at the loop regions. Plasmid-coded Rom protein stabilizes the product of the interaction. In this paper, the mechanism of the loop-to-loop interaction between pairs of RNA stem-loops having various nucleotide sequences is studied. Binding of two stem-loops containing six to eight nucleotides in their loops requires that the loop sequences be complementary, whereas the stem sequences need not be. The association rate constants for binding of complementary pairs with various sequences are relatively similar, around 1 x 10(6) M-1 S-1. On the other hand, the rates of dissociation of the complexes vary greatly depending on the loop sequence, even for complexes having the same base composition, suggesting a strong effect of base-stacking. All the complementary bases in the seven-nucleotide loops participate in complex formation, and the resulting complex is bent a little at the interacting region. Rom binds and stabilizes any complex formed by pairs containing fully complementary loop sequences. Structures are proposed for the RNA complexes with and without Rom.

Quantitation of ColE1-encoded replication elements.

Replication of the Escherichia coli plasmid ColE1 initiates from an RNA primer. This primer is formed by a ColE1 RNA II molecule that remains hybridized to its DNA template in the origin region after transcription. Continued hybridization is inhibited by prior binding to RNA II of another ColE1 transcript, RNA I; and this interaction is regulated by the plasmid-encoded Rom protein. To understand the quantitative aspects of regulation of ColE1 synthesis, we have measured the levels of RNA I, RNA II, and Rom protein in vivo, as well as the half-lives of the RNAs. The intracellular concentrations of RNA I, RNA II, and Rom protein were found to be about 1 microM, 7 nM, and 1 microM, respectively; and the RNAs had half-lives of about 2 min. A simple model derived from these results indicates that the plasmid copy number is little affected by the rate of RNA II synthesis but is strongly dependent on that of RNA I.

A mechanism of formation of a persistent hybrid between elongating RNA and template DNA.

At the replication origin of ColE1 plasmid, a persistent hybrid is formed between the primer precursor (RNA II) and its template DNA. The wild-type sequence in the region 13 to 20 bp upstream (-20 region) of the origin is required to form this persistent hybrid. While the template strand for transcription of this region, containing a stretch of six dC residues, is needed, the nontemplate strand can be deleted. Certain mutations in far upstream regions that prevent hybrid formation are suppressed by the nontemplate strand deletion. In RNA II that is forming a persistent hybrid, the region about 265 nucleotides upstream of the origin (-265 region) can also form a hybrid with the template DNA. The -265 region of RNA II that consists of a stretch of six rG residues probably interacts with the dC stretch of the -20 region in the template strand to promote hybrid formation.

Control of ColE1 plasmid replication. Intermediates in the binding of RNA I and RNA II.

Replication of plasmid ColE1 is regulated by a plasmid-specified small RNA (RNA I). RNA I binds to the precursor (RNA II) of the primer for DNA synthesis and inhibits primer formation. The process of binding of RNA I to RNA II that results in formation of a stably bound complex consists of a series of reactions forming complexes differing in the stability. Formation of a very unstable early intermediate that was previously inferred from the inhibition of stable binding caused by a second RNA I species was firmly established by more extensive studies. This complex is converted to a more stable yet reversible complex that was identified by its RNase sensitivity, which was altered from that of the earlier complex or from that of free RNA I or RNA II. In these complexes, most loops of RNA II interact with their complementary loops of RNA I. The kinetic and structural analyses of the binding process predict formation of a complex interacting at a single pair of complementary loops that precedes formation of these complexes. Thus the process of binding of RNA I to RNA II is seen to consist of a sequence of reactions producing a series of progressively more stable intermediates leading to the final product.

Control of ColE1 plasmid replication. Interaction of Rom protein with an unstable complex formed by RNA I and RNA II.

A transcript (RNA I) from ColE1 inhibits initiation of replication of the plasmid DNA by binding to the precursor of the primer RNA (RNA II). The ability of RNA I to inhibit replication is altered by the presence of a plasmid-specified small protein, Rom. In vitro, RNA I binds to RNA II to form a very unstable complex, C*. Binding of a single molecule of Rom converts C* to a more stable complex, Cm*. Each of these complexes, C* or Cm*, transforms to a more stable complex, C** or Cm**, respectively. While formation of complex C* or Cm* is inferred from the inhibition of binding caused by a second RNA I species, that of complex C** or Cm** is detected by alteration of RNase sensitivity. Complex C* converts to complex Cm* very rapidly upon addition of Rom to the medium and complex Cm* converts to complex C* very rapidly by removal of Rom from the medium. On the other hand, complexes C** and Cm** do not rapidly interconvert, but can eventually transform to the same stable final product. Thus, Rom affects binding of RNA I to RNA II through conversion of a very unstable early intermediate to a more stable complex, creating a second pathway for their stable binding.

Complex formed by complementary RNA stem-loops and its stabilization by a protein: function of CoIE1 Rom protein.

A small plasmid-specified RNA (RNA I) inhibits formation of the RNA primer for CoIE1 DNA replication by binding to its precursor (RNA II). Binding is modulated by the plasmid-specified Rom protein. Both in the presence and absence of Rom, binding starts with interaction between loops of RNAs. To understand the mechanism of binding, we examined the interactions of pairs of single stem-loops that are complementary fragments of RNA I and RNA II. We found that these complementary single stem-loops bind to each other at their loops, forming an RNAase V1-sensitive structure. Rom protects the complex from cleavage and from alkylation of phosphate groups by ethyinitrosourea. A single dimer of Rom binds to the complex by recognizing the structure rather than its exact nucleotide sequence. Rom enhances complex formation by decreasing the rate of dissociation of the complex. Structures of RNA complexes formed in the presence and absence of Rom are proposed.

Rom transcript of plasmid ColE1.

The ColE1 Rom protein contributes to copy number control by affecting the rate of formation of a complex between RNA II, the precursor of the primer for DNA replication, and RNA I, a small RNA complementary to the 5' end of RNA II. Interaction of RNA I with RNA II can affect plasmid copy number by preventing primer formation. Although the RNA I and RNA II transcripts have been well characterized, the rom mRNA has not previously been detected. We have now identified the rom mRNA, and determined its start site both in vitro and in vivo. We also have found that the rom mRNA terminates at any of several sites, and that its synthesis is not autoregulated by the Rom protein. By using an internal standard RNA to estimate the efficiency of detection of the rom mRNA, its level was determined to be about 1 molecule per cell.

Organization of the early region of bacteriophage phi 80. Genes and proteins.

An EcoRI segment containing the early region of bacteriophage phi 80 DNA that controls immunity and lytic growth was identified as a segment whose presence on a plasmid prevented growth of infecting phi 80cI phage. The nucleotide sequence of the segment (EcoRI-F) and adjacent regions was determined. Based on the positions of amber mutations and the sizes of some gene products, the reading frames for five genes were identified. From the relative locations of these genes in the genome, the properties of some isolated gene products, and the analysis of the structures of predicted proteins, the following phi 80 to lambda analogies are deduced: genes cI and cII to their lambda namesakes; gene 30 to cro; gene 15 to O; and gene 14 to P. An amber mutation by which gene 16 was defined is a nonsense mutation in the frame for gene 15 protein, excluding the presence of gene 16. An amber mutation in gene 14 or 15 inhibits phage DNA synthesis, as is the case with their lambda analogues, gene O or P. Some characteristics of proteins from the early region predicted from their primary structures and their possible functions are discussed.

Transcriptional regulation of early functions of bacteriophage phi 80.

To study the expression of early functions of phi 80 phage, various segments from the early region were transcribed with RNA polymerase. Two major transcripts (from promoters PL and PR) whose synthesis was inhibited by the CI protein were identified. Synthesis of the third major transcript (from promoter PRM) was induced by the CI protein. These studies define two operator-promoter regions, OLPL and ORPRPRM. This mode of transcription from the early region is similar to that of phage lambda. However, there are the following major differences. One is the presence of a p-independent terminator of transcription from promoter PL located immediately after gene N and the absence of a p-dependent terminator that corresponds to tR1 of lambda. The other is the uniqueness of the structure and function of the operators. Both OL and OR operator regions consist of three sites, each containing a highly homologous 19 base-pair sequence. In each site, consensus octanucleotide sequences (half-sites) exhibit dyad symmetry, except in one of the sites where the sequences are arranged tandemly. In addition, each operator region also contains a single half-site. The modes of binding of the CI protein and gene 30 protein to these operator sites are quite different from those of the lambda proteins to the lambda operators. For example, binding of the phi 80 CI protein to the OR1 site is less tight than its binding to the OR2 or OR3 site. The gene 30 protein binds to the OR1 site as tightly as to the OR3 site.

Multiple mechanisms for initiation of ColE1 DNA replication: DNA synthesis in the presence and absence of ribonuclease H.

A transcript (RNA II) of plasmid ColE1 that hybridizes with the template DNA is cleaved by RNAase H and used as a primer by DNA polymerase I. However, the plasmid can replicate in bacteria lacking both enzymes, apparently using a different mechanism of initiation of replication. Here we report in vivo and in vitro studies on initiation of DNA replication in the presence or absence of either or both enzymes. Hybridization of RNA II with the template DNA is always required for initiation. Hybridized RNA II is cleaved by RNAase H to form a primer or used as a primer without cleavage by RNAase H. Hybridization also creates a single-stranded region on the nontranscribed strand that can serve as a template for synthesis of the lagging strand in a reaction that does not require DNA polymerase I. Lagging strand synthesis terminates 17 nucleotides upstream of the normal replication origin, forcing unidirectional replication.

Transcriptional activation of ColE1 DNA synthesis by displacement of the nontranscribed strand.

Plasmid ColE1 can replicate using RNAase H and DNA polymerase I. However, it can also replicate in the absence of these enzymes. In this case, formation of a persistent hybrid between a transcript (RNA II) and the DNA indirectly activates subsequent DNA synthesis, instead of providing a primer as it does in the presence of these enzymes. To activate DNA synthesis, a certain length is required for the hybridized region and the region of minimum length cannot include a palindrome. These results show that the single-stranded region of DNA displaced by the hybridization is responsible for the activation. A single-stranded region was identified on the nontranscribed strand by its enhanced reactivity to dimethyl sulfate. The necessary length for the single-stranded region is at least 40 nucleotides. The region probably provides a site for initial binding of a helicase that further unwinds the template DNA for initiation of DNA synthesis.

Factor-independent termination of transcription in a stretch of deoxyadenosine residues in the template DNA.

The primer transcript of plasmid ColE1 extends beyond the replication origin in either of two different modes. It does or does not form a hybrid with the template DNA. When a stretch of 20 deoxyadenosine residues is inserted into the template strand downstream of the origin, more than 90% of hybridized transcripts and about 10% of unhybridized transcripts terminate at the insert. When the number of inserted residues is reduced to ten, the corresponding values are decreased considerably, while the sites of termination are almost unchanged. A palindrome immediately before the stretch increased the efficiency of termination of unhybridized transcripts. Upon termination of hybridized transcripts, RNA polymerase detaches from the template. A structure made by a hybrid or by folded RNA may affect a property of transcription, and a stretch of deoxyadenosine residues including those beyond the actual site of termination may facilitate detachment of RNA polymerase from the template DNA.

Control of ColE1 plasmid replication: binding of RNA I to RNA II and inhibition of primer formation.

Formation of the primer for ColE1 DNA replication from the primary transcript, RNA II, is regulated by an antisense transcript, RNA I. Exposure of elongating RNA II 100-360 nucleotides long to RNA I inhibits subsequent primer formation. However, primer forms normally for transcripts longer than 360 nucleotides. Therefore, the rate of binding of RNA I to RNA II is crucial to regulation. The binding rate varies among RNA II transcripts of different lengths. Transcripts longer than 200 nucleotides are bound faster than shorter ones. The Rom protein enhances binding of RNA I to these longer transcripts, whereas it suppresses binding to some shorter ones. The insensitivity to RNA I of transcripts longer than 360 nucleotides is not due to the absence of RNA I binding but to this binding having no effect on primer formation.