Oligonucleotide dendrimers: stable nano-structures.

DNA dendrimers with two, three, six, nine or 27 arms were reassociated as complementary pairs in solution or with an array of complementary oligonucleotides on a solid support. In all cases, duplex stabilities were greater than those of unbranched molecules of equal length. A theoretical treatment for the process of dissociation of dendrimers explains the major properties of the complexes. The favourable features of DNA dendrimers-their enhanced stability and the simple predictability of their association behaviour-makes them promising as building blocks for the 'bottom up' approach to nano-assembly. These features also suggest applications in oligonucleotide array/DNA chip technology when higher hybridisation temperatures are required, for example, to melt secon-dary structure in the target.

Arrays of complementary oligonucleotides for analysing the hybridisation behaviour of nucleic acids.

Arrays of oligonucleotides corresponding to a full set of complements of a known sequence can be made in a single series of base couplings in which each base in the complement is added in turn. Coupling is carried out on the surface of a solid support such as a glass plate, using a device which applies reagents in a defined area. The device is displaced by a fixed movement after each coupling reaction so that consecutive couplings overlap only a portion of previous ones. The shape and size of the device and the amount by which it is displaced at each step determines the length of the oligonucleotides. Certain shapes create arrays of oligonucleotides from mononucleotides up to a given length in a single series of couplings. The array is used in a hybridisation reaction to a labelled target sequence, and shows the hybridisation behaviour of every oligonucleotide in the target sequence with its complement in the array. Applications include sequence comparison to test for mutation, analysis of secondary structure, and optimisation of PCR primer and antisense oligonucleotide design.

Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models.

An efficient method was developed for making complete sets of oligonucleotides of defined length, covalently attached to the surface of a glass plate, by synthesizing them in situ. A device carrying all octapurine sequences was used to explore factors affecting molecular hybridization of the tethered oligonucleotides, to develop computer-aided methods for analyzing the data, and to test the feasibility of using the method for sequence analysis. Further development is needed before the method can be used routinely, but our work shows that it has a number of potential advantages over gel-based methods: it should be easy to automate; the quality of the sequence results can be evaluated statistically; it provides a powerful way of comparing related sequences and detecting mutation; it can be applied to both DNA and RNA; and specific motifs can be incorporated into all sequences of the array to focus analysis on sequences of biological interest.

DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts.

Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control. We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system. Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2 microns replication origin. 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500-1000 bp). Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule. Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo.

Maximum entropy image reconstruction of DNA sequencing gel autoradiographs.

DNA sequencing gel autoradiographs become increasingly difficult to read as one moves up the gel, due to crowding and overlapping of the bands. Maximum entropy image reconstructions of the autoradiograph improve the ease with which crowded sequence data can be read, and extend the region of the gel in which reading is possible. Superior reconstructions are obtained by using nonuniform models based on the initial reconstructions.

Clones from an 840-kb fragment containing the 5' region of the DMD locus enriched by pulsed field gel electrophoresis.

Detailed analysis of a large region of genomic DNA is facilitated by generating overlapping clones covering the entire region. These clones are usually obtained by bidirectional "walking" using either bacteriophage lambda or cosmid cloning vectors. This is a slow procedure when starting from a single start site. Multiple start sites are an advantage, and here we describe a method of generating clones from an extensive region of the Duchenne muscular dystrophy locus by preparative pulsed field gel electrophoresis using the chromosome of interest isolated in a cell hybrid. We have generated 12 clones mapping to an 840-kb SfiI fragment of DNA from the Xp2.1 region of the X chromosome, where the DMD gene has been localized. Further localization of these clones to the four subregions of the 840-kb fragment indicates that the clones are distributed throughout the fragment. The feasibility of using this approach to generate probes close to other loci is discussed.

Automatic reading of DNA sequencing gel autoradiographs using a large format digital scanner.

We have developed a large format digital scanner for several applications in nucleic acid analysis. Here we describe the scanning of autoradiographs of DNA sequencing gels and a set of programs for reading the base sequence. The programs correct distortions in the gel, recognize bands by their characteristic shape and assign bases to bands by weighting band position and intensity. The sequence read in this way is as accurate as that read by an expert.

On the mechanism of amplification of satellite II DNA sequences of the domestic goat Capra hircus.

A restriction enzyme analysis of the satellite II DNAs of the domestic goat Capra hircus, sheep Ovis aries and ox Bos taurus (p = 1.720, 1.723 and 1.722 g/cm3, respectively) has been carried out and shows that, although all three are composed of repeat units of 700 base-pairs, goat satellite II is present in the genome primarily in the form of 2100 base-pair trimers. Unequal crossing-over between repeat units has produced an oligomer series, whose oligomers gradually decrease in copy number the further they are in molecular weight from the trimer. The trimer population is much more uniform than the monomer population, as most trimers have similar restriction patterns, whereas their component monomers differ considerably in their restriction properties. This heterogeneity was confirmed by cross-hybridization of the different monomers of a cloned trimer. Here, heterologous hybrids were much less stable than the homologous hybrids. Attempts were made to simulate such an oligomer series by computer, using a longitudinal (saltatory), and two horizontal (unequal crossover and drive) models. Simulations of both the saltatory and drive mechanisms could produce the oligomer series in approximately the observed ratios, but only the former could simultaneously produce other restriction properties of this sequence family. This was because the other restriction sites were in a different (monomer) register, and it is difficult for a drive model promoting traits in only one register to fix properties in different registers. The unequal crossover model proposed by Smith (1973, 1976) generally produced homogeneous arrays from heterogeneous arrays, but higher-order repeat structures could be produced when the efficiency of crossing-over between different monomer types was reduced. In most of these arrays, the dimer was the predominant oligomer, but in approximately 10% the trimer was predominant. Since the unequal crossover model produces dimeric arrays with high frequency given appropriate conditions, it is an attractive model for explaining the production of satellite DNAs whose structure has evolved through a series of doublings, such as mouse major satellite DNA and the "alphoid" satellite sequences of primates. Other factors necessary for this model to work are generally considered to be natural components of the speciation process. It is therefore suggested that, although the saltatory model conforms most closely to the observed structure of goat satellite II, this particular satellite DNA may represent one of the few cases when the unequal crossover mechanism does not give rise to a dimeric structure.

The significance of leptospiral titres associated with bovine abortion.

To investigate relationships between serological titres to 2 serovars, pomona (L. pomona) and hardjo (L. hardjo), of Leptospira interrogans and abortions, log linear and logit models were fitted to herd and individual cow data from cattle serologically negative for brucellosis. Serological titres to both serovars were significantly related to abortions in individual cows, with L. pomona having a stronger relationship than L. hardjo. L. hardjo was not significant when herd data were analysed. Differences between dairy and beef cattle in the serological titres found to both L. pomona and L. hardjo were detected when data sets of all cattle or cattle with no history of abortion were analysed. The beef/dairy differences may be due to different management practices and/or to different geographical distributions of both serovars and populations of beef and dairy cattle. If there are no cattle in a herd with a reciprocal titre of 3000 or greater for L. pomona, it is unlikely that L. pomona is associated with the abortion problem. There was no specific L. hardjo titre which separated high and low probabilities that the serum came from a cow or herd with an abortion history.

Changes in cattle tick control practices in south eastern Queensland from 1977 to 1982.

Results of a survey of cattle producers in south eastern Queensland in 1982 concerning cattle tick control have been compared with those of a similar survey carried out in 1977-78. During the interval between these surveys the proportion of beef farms with tick resistant (Bos indicus infusion) cattle as the main breed had increased from 47.8% to 60.0%. Chemical control methods had improved but many producers were continuing to treat their cattle more often than necessary. Over the period there was an increase in the proportion of farms with small numbers of animals, and 30% of the farms had less than 50 cattle.

Karyotyping and identification of human chromosome polymorphisms by single fluorochrome flow cytometry.

Frequency distributions of fluorescence intensity of ethidium bromide stained human chromosomes from nine phenotypically normal males are cross correlated and autocorrelated following repeated flow cytometric measurements. It is shown that each individual donor produces a fluorescence profile which is both visually and numerically different from those of other individuals in the set. The wide variety of chromosome heteromorphisms which occur to varying degrees for chromosomes 1, 9, 13, 14, 15, 16, 21, 22 and Y give rise to the uniqueness of a given fluorescence profile. Estimates of chromosome heteromorphisms for each individual in the set were made and then compared with parallel results obtained from inspection of Q-banded and C-banded conventional metaphase preparations. Fluorescence profiles identifiable with each individual were also obtained for Hoechst 33258 stained chromosomes.

Detecting radiation damage to human chromosomes by flow cytometry.

The flow karyotype profile of ethidium bromide-stained chromosomes from human peripheral blood lymphocytes has been analysed following exposure of lymphocytes to graded series of X-ray doses in vitro. Flow analysis offers the potential for rapid counting of chromosome abnormalities and it is shown that the level of background fluorescence, the distribution of fluorescence and the area of peaks associated with the larger chromosomes, are altered in a dose-related fashion following previous exposures of cultured lymphocytes to 50-400 rad. Moreover, parallel manual analysis of the incidence of chromosome aberrations in metaphase samples of the irradiated cells show a close correlation between flow karyotype profile distortion and aberration frequency. It is estimated that for any given irradiated blood sample doses above 100 rad could be detected with certainty.

Measurement of DNA length by gel electrophoresis. I. Improved accuracy of mobility measurements using a digital microdensitometer and computer processing.

The electrophoretic mobilities of DNA polymer fragments in an agarose gel have been measured from a photograph of the gel by different methods and converted to lengths by the reciprocal method. The method of measurement can introduce large errors in the length estimates. The use of a digital microdensitometer to obtain optical density profiles of gel tracks with subsequent computer processing to find peak positions was found to give the most accurate DNA lengths.

Measurement of DNA length by gel electrophoresis II: Comparison of methods for relating mobility to fragment length.

Relationships between DNA length and electrophoretic mobility in an agarose gel have been compared by estimating the lengths of known DNA polymer fragments, using other fragments in the series as standards. Global estimates were made using 10 fragments as standards; local estimates were made using the two closest fragments on either side of the unknown as standards. Most relationships were fitted by least squares. All the relationships gave more accurate local than global estimates. The most accurate results were obtained using the reciprocal relationship, where the maximum error in the local estimates was less than 0.1%. The semilog relationship gave the least accurate results, with a maximum error in the local estimates of almost 5%. The polymer fragments were also used as standards to estimate the lengths of lambda DNA restriction fragments. Here the estimates were in error by up to 3%, indicating the influence of base composition and sequence on electrophoretic mobility.

Clonidine-induced hyperphagia in monkeys: evidence for alpha 2-noradrenergic receptor mediation.

The mechanism of clonidine-induced hyperphagia and weight gain in monkeys was studied in 11 Stumptail macaques. Clonidine induced a significant increase in food intake over baseline levels and a significant weight gain after the 3-day treatment period. Both changes induced by clonidine were antagonized by the alpha 2-noradrenergic antagonist yohimbine, but not by prazosin, an alpha 1-noradrenergic antagonist. These results suggest that clonidine-induced hyperphagia and weight gain in monkeys are mediated through alpha 2-noradrenergic receptors.

A survey concerning cattle tick control in Queensland. 2. Managerial aspects which indirectly affect tick control.

This paper reports those findings concerning property management from the 1977-78 survey of tick control in Queensland, which could effect efficient tick control and also presents a profile of the cattle industry in the tick infested areas at the time of the survey. The average property size was 400 hectares and the average number of cattle on the properties was 726.4. Larger properties tended to have crossbred Zebu cattle. The average number of paddocks per property was 7.2. The most popular stabilised crossbred Zebu breed was Santa Gertrudis but the most common type of cattle was crossbred Zebu, which was on 30% of properties. There were negligible numbers of crossbreed Zebu cattle on dairy properties. Control of other external parasites may result in additional dippings in areas where they occur. Although fence maintenance appears to be carried out regularly it is clear that the effectivness of tick control measures, such as pasture spelling and dipping which depend on clean musters, could be affected by the occurrence of fire, floods and other natural disasters on half the properties in the tick infested areas. There is no evidence from results in the survey that attention to other farming activities (mainly cropping) prevents producers from paying sufficient attention to tick control. In fact cropping often provides the opportunity for producers to rotate cattle around pastures. Producers appear to consider the cost of tick control is a burden and they expect assistance from the Government to reduce these costs. They do not appear to have considered how they can reduce the costs themselves.