Theory of linear dichroism of DNA and DNA-containing structures

Analysis has been made of the relationship between the dichroic ratio of DNA polymers and the orientation of their transition dipoles. The molecules may be aligned in a mechanical shear or electric field. The effect of the shear or electric field on the orientation factor has been shown. The results of dipole moments and their dependence on the strength of the electric field have been discussed. Higher orders of coiling, flat packing and their various combinations cause the optical and electrical dichroic ratio to change characteristically, so that the electric and optical anisotropy of DNA can be utilized to study the DNA arrangement in DNA-containing structures.     

Molecular arrangement of DNA in bacteriophage T4

The degree of preferred orientation and the coiling of the deoxyribonucleic acid within phage T₄ was studied by two independent techniques, namely, polarization of fluorescence and uv linear dichroism. A correlation between the two kinds of data was obtained, which indicated that a significant proportion (about 30%) of total phage DNA is aligned preferentially along the long axis of phage heads. Analyses of the data suggest that all of the phage DNA cannot be in a highly supercoiled helical configuration. A few models of the DNA arrangement in T₄ have been discussed in which linear sidewise packings of DNA would be predominant and may explain the observed longitudinal orientation of intraphage DNA.     

Identification of the lampbrush chromosome loops which transcribe 5S ribosomal RNA in Notophthalmus (Triturus) viridescens

The loops which transcribe 5S ribosomal RNA in lampbrush chromosomes of the newt, Notophthalmus (Triturus) viridescens, were identified by hybridizing purified 5S DNA to nascent 5S RNA in situ. The genes which code for 5S RNA were found near the centromeres of chromosomes 1, 2, 6, and 7 by hybridizing iodinated 5S RNA to denatured lampbrush and mitotic chromosomes in situ. These genes and their intervening spacer DNA were isolated from Xenopus laevis using sequential silver-cesium sulfate equilibrium centrifugations. This purified 5S DNA was iodinated and hybridized to non-denatured lampbrush chromosomes in situ, where it bound to nascent 5S RNA on loops at the base of the centromeres of chromosomes 1, 2, 6, and 7. The number of 5S genes present in the haploid chromosome complement of N. viridescens was determined. — The 5S loops were chosen for study, since (1) the synthesis of 5S RNA has been demonstrated during the lampbrush stage, (2) both 5S RNA and 5S DNA could be isolated in pure form, and (3) the localization of the repetitive 5S genes could be verified by conventional in situ hybridization procedures. These methods may be applicable to the identification of other loops, leading to a better understanding of lampbrush chromosome function.     

Ultraviolet dichroic ratio of DNA from T2 and T5 bacteriophages

The dichroic ratios of T5st-O and T2H bacteriophage DNA molecules were measured throughout the ultraviolet region of the spectrum. Two methods of DNA orientation were studied: (1) orientation in solution in a Shimadzu flow dichroism instrument attached to a Beckman DU spectrophotometer, and (2) alcohol precipitation of the DNA from solution and orientation in a thin film on the quartz face of a humidity chamber. Spectra in the latter case were recorded using a Gary Model 14 spectrophotomcter fitted with Glan prisms. The lower wavelength limit was 215 mμ in both systems. The DNA preparations were carefully characterized as to spectral purity, sedimentation coefficient, hyperchromicity, protein content, and DNA content. In addition, the structure of the DNA oriented in films was inferred from x-ray diffraction patterns of fibers of the precipitated DXA. The A and B configurations of DNA in films could not be distinguished by the dichroic ratio measuiements. The dichroic ratio obtained for the film-oriented DNA at high relative humidity shows the same wavelength dependence as for the flow-oriented DNA. The same wavelength dependence for DNA in the fibrous state and in solution, when considered together with the x-ray diffract ion results, indicates that DNA in solution maintains an orientation of bases which is similar to that in fibers. I¹Or both solutions and films of DNA, the dichroic ratio is constant from 290mμ to 240 mμ and increases at wavelengths below 240 mμ. The increased parallel absorption below 240 mμ is consistent with the existence of an n→π* transition. The inherent molecular dichroic ratio is found to be the same for T5st-O DNA and T2H DNA in solution, and is a maximum of 0.09 ± 0.02 at 260 mμ.     

Observations on the cytology of Bipes (Amphisbaenia) with special reference to its lampbrush chromosomes

The positions and general anatomical and histological characteristics of the gonads of Bipes biporus and B. canaliculatus are described. The amounts of DNA per haploid chromosome set have been measured in both species, the values being 1.83 and 2.0 pg for biporus and canaliculatus respectively. The karyotypes of both species are described on the basis of data from mitotic and meiotic metaphase chromosome sets and from lampbrush chromosomes. B. biporus has 10 macrochromosomes and 11 microchromosomes. B. canaliculatus has 11 macrochromosomes and 11 microchromosomes. The karyotypes of the two species differ distinctly with regard to the shapes of 3 of the macrochromosomes. Chiasma distribution is described for male meiosis in B. biporus. Studies of the lampbrush chromosomes of both species show the chiasma distribution in the female to be generally similar to that found in the male biporus. In B. canaliculatus, lampbrush chromosomes with maximally extended lateral loops are found in oocytes that are oblate spheroids measuring 0.7×1.0 mm along their short and long axes respectively, these being well before the start of the major phase of vitellogenesis. Smaller oocytes have more distinct chromomeres and shorter loops. Microchromosomes take the form of typical small lampbrush chromosomes in oocytes. There are at the most 1,000 chromomeres per haploid set of lampbrush chromosomes in B. canaliculatus. Chiasmata are described from lampbrush preparations in which the two half-bivalents are firmly attached to one another without evident association of their axes, indicating the possibility of chiasmate association between the DNA axes of lateral loops. There are remarkably few extrachromosomal nucleoli in Bipes oocytes, and its is suggested that this may indicate a level of ribosomal gene amplification that is much lower than that found in fish and Amphibia. The observations are particularly discussed in relation to current ideas concerning the structure and function of lampbrush chromosomes.     

Ultraviolet Inactivation Dichroic Ratio of Oriented fd Bacteriophage

Polarized UV light irradiation of flow-oriented fd bacteriophage indicates that the degree of damage (inactivation) depends on the relative orientation of the light polarization vector and the plane of the DNA bases. The technique of anisotropic UV inactivation was evaluated, and further information on the orientation in this virus was gained. The fd bacteriophage were aligned and irradiated with plane-polarized monochromatic UV light either parallel or perpendicular to the virus axis. Variation of the inactivation dichroic ratio with wavelength implicated virus inactivation by light absorbed in both the DNA and protein. Analysis of the wavelength variation of inactivation dichroic ratios gave molecular dichroic ratios of 0.76 and 1.48 for the DNA and protein components, respectively. On the basis of these anisotropic inactivation studies, the average angle of DNA base tilt in fd was calculated to be 29-32°, a value in agreement with the absorption dichroism studies of Bendet and Mayfield.     

A theory for polarized fluorescence microscopy of chromosome structures

A theory for the determination of DNA arrangements in DNA-containing specimens, using planar aromatic dye molecules as probes for plane polarization of fluorescence, has been described. At low dye-to-DNA concentrations, the dye molecules are sandwiched between the stacked bases of DNA; hence, the fluorescence from the dye bound to a local region of DNA helix is plane-polarized with the polarization direction perpendicular to the local axis of DNA. The degree of such polarization from an aligned DNA-specimen complexed with dye is determined both by the DNA orientation and the conformational state (e.g., base tilt) of DNA into that specimen. Analysis has been made of the relationship between the degree of polarization and the orientation of the emitting dipoles of dye. The dye complexes may be aligned in a mechanical shear or electric field. However, any change in the orientation distribution of the emitting dipoles due to force fields should be taken into account. With some assumptions and approximations, the magnitude and the direction of maximum polarization can be related to different orders of DNA coiling and to their various combinations. Since the measured polarization is averaged over all DNA regions of the specimen, if the magnitude of polarization is appreciable and the polarization occurs in the specific direction of the specimen, the theory helps to eliminate several probable arrangements of DNA. The predominant molecular features of the actual DNA arrangement can be determined through this process of elimination, as explained in two subsequent papers with T-even bacteriophage and chromosome systems.     

The maps of the lampbrush chromosomes of Triturus (Amphibia urodela)

The lampbrush chromosomes of Triturus vulgaris meridionalis were isolated from the germinal vesicle of medium and large-sized oocytes and studied with phase-contrast microscope. The maps were constructed on the basis of the lengths and major morphological features of the chromosomes. The length of each map is equal to the mean of the relative lengths of the corresponding chromosome from different oocytes (the relative length of each chromosome is represented by the ratio between its absolute length and that of chromosome XII from the same complement, conventionally considered as 100 units long). The maps arranged in decreasing length order, were oriented according to the most frequent position of chiasmata, as centromeres were not always evident. — Chromosomes VI and XI bear a sphere in subterminal position. Landmarks typical for T. vulgaris meridionalis are the loops inserted on chromosomes VIII (47 units), X (23 units), XI (34 units) and XII (34 units) frequently presenting themselves under the form of double loop bridges of considerable extension. On chromosomes I (4 units), VI (13 units), X (4 units) and XI (36 units) giant bodies were found that are sometimes comparable to dense-matrix loops. Chromosome XI includes a nucleolus-organizing region, sometimes identifiable by the presence of an inserted nucleolus. Normal and granular loops (much extended at times), axial granules, globules, and loopless bars supplement the morphology of the lampbrush chromosomes of this species.     

Localization of the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei by fluorescence in situ hybridization

We have used fluorescence in situ hybridization to map the positions of the different repetitive DNA sequences from the region forming the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei. This region harbours a megabase cluster of tandemly organized repeats of the Y-specific ay1 family and a megabase cluster of tandem repeats of the related Y-specific YsI family. In addition, ay1 repeats also occur in short blocks that are interspersed by other repetitive DNA sequences that we call Y-associated, since they have additional copies on other chromosomes. Using specific probes for ay1, YsI and Y-associated DNA sequences, we show that there is one large proximal cluster of YsI repeats and one, more distally located, large cluster of ay1 repeats. The Y-chromosomal copies of the Y-associated sequences are located in the most distal part of the ay1 cluster. This is consistent with the juxtaposition of ay1 and Y-associated sequences in more than 300 kb of cloned genomic DNA. Since both ay1 and Y-associated sequences have been shown to be transcribed in the Nooses, the lampbrush loop is formed in a distal region of the short arm of the Y chromosome, adjacent to the terminally located nucleolus organizer region. The clusters of homogeneous ay1 and YsI repeats are of no functional significance for the formation of the lampbrush loop.     

Structural studies on prolyl hydroxylase. Conformation from circular dichroism spectroscopy

The circular dichroism spectra of purified prolyl hydroxylase [EC 1.14.11.12] in the native and heat-denatured states were obtained at pH 7.8. Analysis of the far-uv spectrum of the native enzyme gave an estimate of 40% alpha-helix, 40% beta-structure and 20% random coil. The near-uv spectrum contained several negative dichroic bands that can be attributed to phenylalanyl, tyrosyl and tryptophyl residues situated in an asymmetric environment in the protein. These bands were not seen in the enzyme denatured by heat. The denaturation was monitored by changes in the alpha-helical content as well as the activity of the enzyme as a function of temperature. The normalized transition profiles with respect to the change in helical content and the loss of enzyme activity were coincidental, indicating the involvement of the alpha-helical segments in maintaining the enzyme activity.     

Evolution of Y chromosomal lampbrush loop DNA sequences of Drosophila

The evolutionary conservation of Y chromosomal DNA sequences of Drosophila hydei in different species of the genus Drosophila was studied by in situ hybridization and on genomic DNA blots of restriction enzyme digested DNA. We demonstrated that Y specific DNA sequences, which form major parts of lampbrush loops related to the male fertility genes, are only retained in a few closely related species during evolution. Other Y chromosomal DNA sequences, also present in lampbrush loops but with homology to autosomal and X chromosomal locations, were found in distant species. We propose a model for the evolution of the Y chromosomal lampbrush loops.     

Higher levels of organization in chromosomes.

On the basis of recent results a unified view of different aspects of the higher levels in the organization of chromatin in chromosomes is presented. Basic to these forms of organization is the arrangement of DNA in the complex with nucleosomes and recent studies suggest that at least some species of satellite DNA may maintain a fixed DNA sequence relationship to the phasing of nucleosomes. Special proteins such as the high-mobility group (HMG) proteins or other non-histone proteins could serve specific functions in the recognition of satellite DNA sequences.In the presence of histone H1 the 110 Å nucleosome fiber formed from the basic string of nucleosomes can be further condensed into a thicker 250–300 Å fiber formed by a solenoidal coiling of the 110 Å fiber with about 6–8 nucleosomes per turn and the available evidence suggests that these structures are found in mitotic chromosomes as well as other forms of inactive chromatin. A further level of coiling of the 250–300 Å solenoid has been suggested by our recent studies of disintegrated mitotic chromosomes consisting of a thin-walled tube with an outer diameter of 4000 Å referred to as the unit fiber. This structure would account for a factor of 1400 × contraction of DNA in mitotic chromosomes which in their intact state are only 5-fold more contracted. The recently described “scaffold” proteins could be responsible for this final coiling of the unit fibers in intact chromosomes.Meiotic chromosomes are generally less contracted than mitotic chromosomes. An extreme example of this are lampbrush chromosomes that apart from the axial segments which might contain some structural proteins appear to consist of naked DNA arranged in lateral loops. In the later stages of meiosis more condensed structures arise as exemplified by the synaptonemal complex during the pachytene stage in many organisms. The characteristic features of this structure are interpreted to suggest that the structure consists of lateral components containing two parallel 110 Å nucleosome fibers each representing the axial segments of two sister chromatids. From these paired regions loops protrude laterally in a manner which closely resembles the less condensed lampbrush chromosomes. The implication of this structure in the process of crossingover is discussed.Less is known about the organization of chromatin in interphase nuclei, but structures analogous to the loop-like structures in meiotic chromosomes are suggested on the basis of the isolation of supercoiled DNA loops constrained by RNA-DNA and protein-DNA interactions. The position of these loops is suggested to be fixed by specific repeated DNA sequences that could be associated with specific tenacious non-histone or HMG proteins.     

Electric birefringence of poly-L-lysine hydrobromide in methanol-water mixtures and helix-coil transition induced by high electric fields

The electric birefringence of poly-L -lysine hydrobromide in methanol–water mixtures has been measured at 25 °C over a wide range of field strengths by use of the rectangular pulse technique. An abrupt change in the specific Kerr constant was observed between 87 and 90 vol % methanol, corresponding to the solvent-induced helix–coil transition. The specific Kerr constant increased rapidly with dilution in the random coil form, and more slowly in the helical conformation. The field strength dependence of the bire fringence at various concentrations, for both the helical and coil conformations, can be described by a common orientation function, which resembles the theoretical one for the case of permanent dipole moment orientation. This is interpreted in terms of the saturation of ion–atmosphere polarization. The optical anisotropy for the helical conformation was much larger than that for the coil form. Anomalous birefringence signals were observed above a critical field strength (about 5 kV/cm) in 90 vol % methanol. The birefringence passed through a maximum and began to decrease slowly before the pulse terminated, reaching a steady-state value. This steady-state value was closer to that of the coil in the coil in the limit of very high fields. The results indicate that a transition from the charged helix to the charged coil is induced by high electric fields in the transition region. This effect can be explained on the basis of the polarization mechanism proposed by Neumann and Katchalasky.