Determination of Melting Sequences in DNA and DNA-Protein Complexes by Difference Spectra

A graphical formula is presented for determining the base ratio of melted DNA. By use of this formula, the composition of sequences which melt in different portions of the melting curves of Clostridium DNA, Escherichia coli DNA, and mouse DNA were determined. As the DNA melts, the per cent of adenine and thymine (AT) in the melted sequences decreases linearly with temperature. The average composition of sequences which melt in a given part of the melting curve is proportional to the base ratio of the DNA. The concentration and average composition of sequences were determined for three parts of the melting curves of the DNA samples, and a frequency distribution curve was constructed. The curve is symmetrical and has a maximum at about 56% AT. The distribution of GC-rich sequences on the E. coli chromosome was estimated by shearing, partially melting, and fractionating the DNA on hydroxylapatite. GC-rich sequences appear to occur every thousand base pairs, and have a maximum length of about 180 base pairs. The graphical formula was applied to the determination of the composition of sequences which melt in different parts of the melting curve of chromatin. Throughout the melting curve, the composition of the melting sequences is about 60% AT, which appears to suggest that relatively long sequences are melting simultaneously. Their melting temperature may be a function of the composition of the protein on different parts of the DNA. The problem of light scattering in DNA-protein and DNA was also investigated. A formula is presented which corrects for light scattering by relating the intensity of the scattered light to the rate of change of absorbance of DNA with wavelength.     

Methods for Detecting Food-borne Enteroviruses

A method previously reported for detecting virus in a model system composed of cottage cheese contaminated with coxsackievirus type A9 has been adapted to detecting selected strains of enteroviruses in a variety of foods. Bentonite is omitted and serum is added for extracting virus from low-protein foods. Samples of foods, usually 25 g, must contain at least 3 to 4 plaque-forming units for a 50% probability of detecting virus. Sensitivity in detecting echovirus type 6 was lower than that for the other viruses used. After extraction from potato salad, poliovirus type 2 was completely reactivated if it had been neutralized with coproantibody, but it was only partially reactivated if neutralized with hyperimmune rabbit serum.     

Der Einfluß des Zeugungsalters auf die Mutationen zu Hämophilie A

Assuming that in sibships with sporadic hemophilia the mothers of the patients are already carriers the relationship between the age of the mother's father at birth of the mother and frequency of hemophilia among his grandsons was examined.The ages of the mother's father at birth of the mother of 40 patients with sporadic hemophilia A was compared with that of a control-group as well as with the ages of the mother's fathers at birth of the mothers of patients from families where hemophilia A is inherited. The mean age of these grandfathers was found to be increased. Using the Mann-Whitney-U-test for comparing the ages of grandfathers of sporadic cases with the control-group there is to be found P=.0025, which is highly significant statistically. Comparing data from sibships with sporadic hemophilia with data from sibships where hemophilia is inherited there is no significant difference — perhaps according to the small number of inherited cases (19, P=0.065) —, but the deviation is in the same direction. Comparison of data both from the inherited cases and the control-group with data of sporadic cases gives P=.0087.There is perhaps a connection between parental age and number of children, but it is shown to have no important influence in our material. On the other hand fathers with a higher number of children are significant more frequent among the grandfathers than among the controls. This difference cannot be explained sufficiently. Between cases of sporadic and inherited hemophilia there is no clear cut difference.Certainly there exists a relationship between parental age and birth-rank. Therefore the mothers of sporadic cases — unlike to carriers of sibships with inherited hemophilia — take clearly higher ranks in birth-order than it is theoretically to be expected. Penrose published a method of separating the relative aetiological effects of birth-order and parental age. Using this method an influence of birth-order cannot be found after excluding the influence of parental age. Hence, paternal age seems to be the determining factor.It is discussed which model of mutation the hemophilia-mutation belongs to because of this relationship. We would count it to No. 2 of the classification given by Vogel where mutations are due to copy errors. So cases of sporadic hemophilia seem equal to those of sporadic achondroplasia.

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Diese Arbeit wurde der Medizinischen Fakultät der Universität Hamburg als Inaugural-dissertation vorgelegt.

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Herrn Prof. Dr. F. Vogel danke ich für wertvolle Hinweise, insbesondere zur statistischen Methodik.     

Histochemische Untersuchungen an der Leber nach Verbrennung

Zusammenfassung An männlichen weißen Ratten wurde das Verhalten des Enzymmusters der Leber 72 Std nach Verbrühung untersucht. Es fanden sich Veränderungen im Sinne einer Aktivitätsverminderung bei der NAD-Cytochrom-c-Reduktase, Isocitronensäuredehydrogenase, ATPase und den PJS-positiven Substanzen. Eine Zunahme der Aktivität wurde bei der alkalischen Phosphatase und einigen Esterasen beobachtet, ebenso traten auch die scharlachrotfärbbaren Substanzen auf. Uneinheitliche Reaktionen zeigten die Succinodehydrogenase sowie die Leucinaminopeptidase, unverändert blieben Glukose-6-phosphat-dehydrogenase, -Glucuronidase, saure Phosphatase, die o-Diacetylbenzol- und die DDD-Reaktion.     

DNA encapsidation by viruslike particles assembled in insect cells from the major capsid protein VP1 of B-lymphotropic papovavirus.

Capsids of polyomaviruses--small, nonenveloped DNA viruses--consist of the major structural protein VP1 and the minor structural proteins VP2 and VP3. The contributions of the individual capsid proteins to functions of the viral particle, such as DNA encapsidation, cell receptor attachment, entry, and uncoating, are still not clear. Here we show that viruslike particles assembled in nuclei of insect cells from VP1 of the monkey B-lymphotropic papovavirus (LPV) are sufficient to unspecifically encapsidate DNA. LPV VP1 expressed in large amounts in insect cells by a baculovirus vector assembled spontaneously in the nuclei to form viruslike particles. After metrizamide equilibrium density gradient purification and nuclease digestion, a fraction of these particles was shown to contain VP1-associated linear, double-stranded DNA with a predominant size of 4.5 kb. The fraction of DNA-containing VP1 particles increased with time and dose of baculovirus vector infection. The DNA-containing particles, further purified by sucrose gradient centrifugation, appeared as "full" particles in negative-staining electron microscopy. As shown by DNA hybridization, the encapsidated DNA consisted of insect cell and baculoviral sequences with no apparent strong homology to LPV sequences. Three non-LPV VP1-derived host proteins with apparent molecular masses of approximately 14, 15, and 16 kDa copurified with the DNA-containing particles and may represent insect cell histones encapsidated together with the DNA. A similar species of host DNA was also found in purified LPV wild-type virions. These data suggest that LPV VP1 alone can be sufficient to encapsidate linear DNA in a sequence-independent manner.     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

Sequence divergence in the ORF6 gene of Mycoplasma pneumonia.

The ORF6 gene product of Mycoplasma pneumoniae is involved in a yet-unknown manner in the adhesion of the bacterium to its host cell. Part of the ORF6 gene is a repetitive DNA sequence (RepMP5), about 1,900 bp long. Seven additional similar copies of RepMP5 are dispersed on the genome. In the independently isolated strains M. pneumoniae M129 and FH, the RepMP5 copies residing in the ORF6 gene are not identical. Two conserved regions, ranging from nucleotides 1 to 799 and from nucleotide 1795 to the end of the gene, border a variable region, ranging from nucleotides 800 to 1794. This variable region differs in DNA sequence and by 201 bp. Analysis of RepMP5 copies outside the ORF6 gene showed that both M. pneumoniae M129 and M. pneumoniae FH carry a RepMP5 copy on a 6-kbp EcoRI fragment which has the same DNA sequence as the variable region of RepMP5 in the M. pneumoniae FH ORF6 gene. According to these data, a switch from the M. pneumoniae M129 ORF6 gene to the M. pneumoniae FH ORF6 gene could take place by gene conversion.     

Identification and characterization of hitherto unknown Mycoplasma pneumoniae proteins

Eleven hitherto unknown Mycoplasma pneumoniae proteins were identified and characterized with respect to their size and subcellular location. This was carried out through the construction of in vitro gene fusions between a modified mouse dehydrofolate reductase (dhfr) gene and selected regions (cosmid clones) of the M. pneumoniae genome and expressing them in Escherichia coli. Positive clones were identified using antibodies against specific fractions of M. pneumoniae. The deduced protein sequences of 11 out of 30 clones did not show significant homologies to known proteins in protein databank searches. Monospecific antibodies against these 11 fusion proteins were used to determine the size and cellular location of the corresponding M. pneumoniae proteins by immunoscreening Western blots of SDS-acrylamide gels from M. pneumoniae cell extracts.