A structural model for a homeotic protein-extradenticle-DNA complex accounts for the choice of HOX protein in the heterodimer.

The genes of the homeotic complex (HOX) encode DNA binding homeodomain proteins that control developmental fates by differentially regulating the transcription of downstream target genes. Despite their unique in vivo functions, disparate HOX proteins often bind to very similar DNA sequences in vitro. Thus, a critical question is how HOX proteins select the correct sets of target genes in vivo. The homeodomain proteins encoded by the Drosophila extradenticle gene and its mammalian homologues, the pbx genes, contribute to HOX specificity by cooperatively binding to DNA with HOX proteins. For example, the HOX protein labial cooperatively binds with extradenticle protein to a 20-bp oligonucleotide that is sufficient to direct a labial-like expression pattern in Drosophila embryos. Here we have analyzed the protein-DNA interactions that are important for forming the labial-extradenticle-DNA complex. The data suggest a model in which labial and extradenticle, separated by only 4 bp, bind this DNA as a heterodimer in a head-to-tail orientation. We have confirmed several aspects of this model by characterizing extradenticle-HOX binding to mutant oligonucleotides. Most importantly, mutations in base pairs predicted to contact the HOX N-terminal arm resulted in a change in HOX preference in the heterodimer, from labial to Ultrabithorax. These results demonstrate that extradenticle prefers to bind cooperatively with different HOX proteins depending on subtle differences in the heterodimer binding site.

The effects of sequence context on DNA curvature.

Recent experiments have exposed significant discrepancies between experimental data and predictive models for DNA structure. These results strongly suggest that DNA structural parameters incorporated in the models are not always sufficient to account for the influence of sequence context and of specific ion effects. In an attempt to evaluate these two effects, we have investigated repetitive DNA sequences with the sequence motif GAGAG.CTCTC located in different helical phasing arrangements with respect to poly(A) tracts and GGGCCC.GGGCCC sequence motifs. Methods used are ligase-mediated cyclization and gel mobility experiments along with DNase I cutting and chemical probe studies. The results provide new evidence for curvature in poly(A) tracts. They also show that the sequence context in which bending and flexible sequence elements are found is an important aspect of sequence-dependent DNA conformation. Although dinucleotide models generally have good predictive power, this work demonstrates that in some instances sequence elements larger than the dinucleotide must be taken into account, and hence it provides a starting point for the appropriate modification and refinement of existing structural models for DNA.

Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse.

Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.

In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities.

A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'-A5T4T3G2C1G1'C2'A3A4'T5'-3'. Mutants containing the sequence Leu-18Tyr-15Xaa-14Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed.

Recognition of diverse sequences by class I zinc fingers: asymmetries and indirect effects on specificity in the interaction between CF2II and A+T-rich elements.

The Drosophila CF2II protein, which contains zinc fingers of the Cys2His2 type and recognizes an A+T-rich sequence, behaves in cell culture as an activator of a reporter chloramphenicol acetyltransferase gene. This activity depends on C-terminal but not N-terminal zinc fingers, as does in vitro DNA binding. By site-specific mutagenesis and binding site selection, we define the critical amino acid-base interactions. Mutations of single amino acid residues at the leading edge of the recognition helix are rarely neutral: many result in a slight change in affinity for the ideal DNA target site; some cause major loss of affinity; and others change specificity for as many as two bases in the target site. Compared to zinc fingers that recognize G+C-rich DNA, CF2II fingers appear to bind to A+T-rich DNA in a generally similar manner, but with additional flexibility and amino acid-base interactions. The results illustrate how zinc fingers may be evolving to recognize an unusually diverse set of DNA sequences.

Molecular phylogeny of the extinct ground sloth Mylodon darwinii.

DNA was extracted from the remains of 35 ground sloths from various parts of North and South America. Two specimens of Mylodon darwinii, a species that went extinct at the end of the last glaciation, yielded amplifiable DNA. However, of the total DNA extracted, only approximately 1/1000 originated from the sloth, whereas a substantial part of the remainder was of bacterial and fungal origin. In spite of this, > 1100 bp of sloth mitochondrial rDNA sequences could be reconstructed from short amplification products. Phylogenetic analyses using homologous sequences from all extant edentate groups suggest that Mylodon darwinii was more closely related to the two-toed than the three-toed sloths and, thus, that an arboreal life-style has evolved at least twice among sloths. The divergence of Mylodon and the two-toed sloth furthermore allows a date for the radiation of armadillos, anteaters, and sloths to be estimated. This result shows that the edentates differ from other mammalian orders in that they contain lineages that diverged before the end of the Cretaceous Period.

Cyclic polyamides for recognition in the minor groove of DNA.

Small molecules that specifically bind with high affinity to any designated DNA sequence in the human genome would be useful tools in molecular biology and potentially in human medicine. Simple rules have been developed to rationally alter the sequence specificity of minor groove-binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids. Crescent-shaped polyamides bind as antiparallel dimers with each polyamide making specific contacts with each strand on the floor of the minor groove. Cyclic polyamides have now been synthesized that bind designated DNA sequences at subnanomolar concentrations.

Mutations in the MSH3 gene preferentially lead to deletions within tracts of simple repetitive DNA in Saccharomyces cerevisiae.

Eukaryotic genomes contain tracts of DNA in which a single base or a small number of bases are repeated (microsatellites). Mutations in the yeast DNA mismatch repair genes MSH2, PMS1, and MLH1 increase the frequency of mutations for normal DNA sequences and destabilize microsatellites. Mutations of human homologs of MSH2, PMS1, and MLH1 also cause microsatellite instability and result in certain types of cancer. We find that a mutation in the yeast gene MSH3 that does not substantially affect the rate of spontaneous mutations at several loci increases microsatellite instability about 40-fold, preferentially causing deletions. We suggest that MSH3 has different substrate specificities than the other mismatch repair proteins and that the human MSH3 homolog (MRP1) may be mutated in some tumors with microsatellite instability.

Identification and characterization of putative transposable DNA elements in solanaceous plants and Caenorhabditis elegans.

Several families of putative transposable elements (TrEs) in both solanaceous plants and Caenorhabditis elegans have been identified by screening the DNA data base for inverted repeated domains present in multiple copies in the genome. The elements are localized within intron and flanking regions of many genes. These elements consist of two inverted repeats flanking sequences ranging from 5 bp to > 500 bp. Identification of multiple elements in which sequence conservation includes both the flanking and internal regions implies that these TrEs are capable of duplicative transposition. Two of the elements were identified in promoter regions of the tomato (Lycoperiscon esculentum) polygalacturonase and potato (Solanum tuberosum) Win1 genes. The element in the polygalacturonase promoter spans a known regulatory region. In both cases, ancestral DNA sequences, which represent potential recombination target sequences prior to insertion of the elements, have been cloned from related species. The sequences of the inverted repeated domains in plants and C. elegans show a high degree of phylogenetic conservation. While frequency of the different elements is variable, some are present in very high copy number. A member of a single C. elegans TrE family is observed approximately once every 20 kb in the genome. The abundance of the described TrEs suggests utility in the genomic analysis of these and related organisms.

Transition metal ion activation of DNA binding by the diphtheria tox repressor requires the formation of stable homodimers.

The diphtheria tox repressor (DtxR) is a transition metal ion-dependent regulatory element that controls the expression of diphtheria toxin and several genes involved in the synthesis of siderophores in Corynebacterium diphtheriae. In the presence of transition metal ions apo-DtxR becomes activated and specifically binds to its target DNA sequences. We demonstrate by glutaraldehyde cross-linking that monomeric apo-DtxR is in weak equilibrium with a dimeric form and that upon addition of activating metal ions to the reaction mixture a dimeric complex is stabilized. Addition of the DNA-binding-defective mutant apo-DtxR(delta 1-47) to apo-DtxR in the absence of transition metal ions inhibits conversion of the apo-repressor to its activated DNA-binding form. We also show that the binding of Ni2+ to both apo-DtxR and apo-DtxR(delta 1-47) is cooperative and that upon ion binding there is a conformational change in the environment of the indole ring moiety of Trp-104. For the wild-type repressor the consequences of this conformational change include a shift in equilibrium toward dimer formation and activation of target DNA binding by the repressor. We conclude that the formation of DtxR homodimers is mediated through a protein-protein interaction domain that is also activated on metal ion binding.

An A-DNA triplet code: thermodynamic rules for predicting A- and B-DNA.

The ability to predict macromolecular conformations from sequence and thermodynamic principles has long been coveted but generally has not been achieved. We show that differences in the hydration of DNA surfaces can be used to distinguish between sequences that form A- and B-DNA. From this, a "triplet code" of A-DNA propensities was derived as energetic rules for predicting A-DNA formation. This code correctly predicted > 90% of A- and B-DNA sequences in crystals and correlates with A-DNA formation in solution. Thus, with our previous studies on Z-DNA, we now have a single method to predict the relative stability of sequences in the three standard DNA duplex conformations.

Different cellular backgrounds confer a marked advantage to either mutant or wild-type mitochondrial genomes.

After the introduction of mitochondria with a mixture of mutant and wild-type mitochondrial DNA (mtDNA) into a human rho degree cell line (143B.206), Yoneda et al. [Yoneda, M., Chomyn, A., Martinuzzi, A., Hurko, O. & Attardi, G. (1992) Proc. Natl. Acad. Sci. USA 89, 11164-11168] observed a shift in the proportion of the two mitochondrial genotypes in a number of cybrid clones. In every case where a shift was observed, there was an increase in the proportion of mutant mtDNA. By using the same cell line (143B.206 rho degree), we also generated cybrids that were either stable in their mitochondrial genotype or showed an increase in the proportion of mutant mtDNA. However, temporal analysis of the same mutant mtDNA type in another rho degree cell line revealed a quite distinct outcome. Those clones that showed a change shifted toward higher levels of wild-type rather than mutant mtDNA. These results indicate that the nuclear genetic background of the recipient (rho degree) cell can influence the segregation of mutant and wild-type mitochondrial genomes in cell cybrids.

Basal body-associated DNA: in situ studies in Chlamydomonas reinhardtii.

We have explored the localization of the uni chromosome (LG XIX) of Chlamydomonas reinhardtii using the technique of in situ hybridization. Using standardized methods of cell fixation together with large chromosome-specific probes we have studied the position of uni DNA sequences in metaphase and interphase cells. We find that in dividing cells uni probes identify a condensed metaphase chromosome that shows no specialized orientation. In interphase cells uni hybridization signals occur on the anterior edge of the nucleus at a position where basal bodies are normally associated with the nuclear envelope. These data reveal an underlying spatial organization of uni chromosomal DNA within the interphase nucleus that may be significant in terms of the fact that this chromosome encodes numerous functions affecting basal body and flagellar assembly.

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.

Simple tandem DNA repeats and human genetic disease.

The human genome contains many repeated DNA sequences that vary in complexity of repeating unit from a single nucleotide to a whole gene. The repeat sequences can be widely dispersed or in simple tandem arrays. Arrays of up to 5 or 6 nt are known as simple tandem repeats, and these are widely dispersed and highly polymorphic. Members of one group of the simple tandem repeats, the trinucleotide repeats, can undergo an increase in copy number by a process of dynamic mutation. Dynamic mutations of the CCG trinucleotide give rise to one group of fragile sites on human chromosomes, the rare folate-sensitive group. One member of this group, the fragile X (FRAXA) is responsible for the most common familial form of mental retardation. Another member of the group FRAXE is responsible for a rarer mild form of mental retardation. Similar mutations of AGC repeats give rise to a number of neurological disorders. The expanded repeats are unstable between generations and somatically. The intergenerational instability gives rise to unusual patterns of inheritance--particularly anticipation, the increasing severity and/or earlier age of onset of the disorder in successive generations. Dynamic mutations have been found only in the human species, and possible reasons for this are considered. The mechanism of dynamic mutation is discussed, and a number of observations of simple tandem repeat mutation that could assist in understanding this phenomenon are commented on.