Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes.

Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.

Excision repair in Drosophila. Analysis of strand breaks appearing in DNA of mei-9 mutants following mutagen treatment.

Excision repair of DNA damage has been analyzed in primary and established cell cultures of Drosophila melanogaster. Chemical and enzymatic assays for pyrimidine dimers reveal a strong deficiency in dimer excision from cells which are mutant at the mei-9 locus. Single-strand interruptions, which appear in high molecular weight DNA after ultraviolet irradiation of control cells have been monitored by alkaline elution. The appearance of such breaks is greatly enhanced by inhibitors of DNA synthesis. In mutant mei-9D2 cells, on the other hand, the level of ultraviolet-induced breaks is much reduced and inhibitors fail to potentiate the response. These results imply that the inhibitors cause an accumulation of the transient strand interruptions that normally occur in excision repair by reducing the rate of the resynthesis step. Failure of the mei-9D2 cells to accumulate such intermediates strongly suggests that the initial nicking never occurs in these cells. Confirmatory experiments have also been performed with the alternate mutagen N-acetoxy-N-acetyl-2-aminofluorene.

Isolation and characterization of a photorepair-deficient mutant in Drosophila melanogaster.

A mutation abolishing photorepair has been localized to map position 56.8 centimorgans on the second chromosome of Drosophila melanogaster. Strains homozygous for the phr allele are totally devoid of photorepair and partially deficient in excision repair. Both defects map to the chromosomal region between pr and c. Since a homozygous phr stock exhibits reduced photoreactivation, the corresponding wild-type allele plays a significant role in UV resistance.

mus308 mutants of Drosophila exhibit hypersensitivity to DNA cross-linking agents and are defective in a deoxyribonuclease.

Mutagen-sensitive strains that identify 16 different Drosophila genes have been screened for alterations in DNA metabolic enzymes. A characteristic defect in an acid-active deoxyribonuclease was observed in strains carrying the six available mutant alleles of the mus308 gene. Since that enzyme is detected at normal levels in a mutant strain that is deficient in the previously identified enzymes DNase 1 and DNase 2, it represents a new Drosophila nuclease that is designated Nuclease 3. The mus308 mutants were originally distinguished from all other mutagen-sensitive mutants of Drosophila because they exhibit hypersensitivity to the DNA cross-linking agent nitrogen mustard without expressing a concurrent sensitivity to the monofunctional agent methyl methanesulfonate. Further observations of hypersensitivity to the mutagens trimethylpsoralen, diepoxybutane and cis-platinum now establish a more general sensitivity of these mutants to agents capable of generating DNA cross-links. In spite of the hypersensitivity of the mus308 mutants to DNA cross-linking agents, the initial incision step of DNA cross-link repair is normal in mus308 cells as assayed by the alkaline elution procedure. The Drosophila mus308 mutants show promise of providing a useful model for analogous defects in other organisms including man.

Identification of a second locus in Drosophila melanogaster required for excision repair.

The mus(2)201 locus in Drosophila is defined by two mutant alleles that render homozygous larvae hypersensitive to mutagens. Both alleles confer strong in vivo somatic sensitivity to treatment by methyl methanesulfonate, nitrogen mustard and ultraviolet radiation but only weak hypersensitivity to X-irradiation. Unlike the excision-defective mei-9 mutants identified in previous studies, the mus(2)201 mutants do not affect female fertility and do not appear to influence recombination proficiency or chromosome segregation in female meiocytes.--Three independent biochemical assays reveal that cell cultures derived from embryos homozygous for the mus(2)D1 allele are devoid of detectable excision repair. 1. Such cells quantitatively retain pyrimidine dimers in their DNA for 24 hr following UV exposure. 2. No measurable unscheduled DNA synthesis is induced in mutant cultures by UV treatment. 3. Single-strand DNA breaks, which are associated with normal excision repair after treatment with either UV or N-acetoxy-N-acetyl-2-aminofluorene, are much reduced in these cultures. Mutant cells possess a normal capacity for postreplication repair and the repair of single-strand breaks induced by X-rays.

Pyrimidine dimers in Drosophila chromatin become increasingly accessible after irradiation.

A prokaryotic DNA-repair enzyme has been utilized as a probe for changes in the accessibility of pyrimidine dimers in Drosophila chromatin following UV irradiation. The results demonstrate a rapid cellular response to physiologically relevant doses of radiation which results in at least a 40% increase in accessible dimers. This increase occurs in two incision-deficient mutants which indicates that the excision-repair process, at or beyond the incision step, is not required or responsible for the increase. In the absence of excision the increase in accessibility persists for at least 2 days following irradiation. The observed increase in accessibility is inhibited by both novobiocin and coumermycin. These inhibitors do not inhibit the initial rate of incision, but do reduce dimer excision measured over more extended periods. A pre-incision process is proposed which actively exposes DNA lesions to excision repair. A fraction of the genome is postulated to be accessible without the intervention of that process.

Re-evaluation of excision repair in the mus304, mus306 and mus308 mutants of Drosophila.

The excision repair capacity of the third chromosomal mus mutations of Drosophila has been re-evaluated. A partial deficiency in the excision repair of pyrimidine dimers originally observed in the mus304 mutants is now attributed to the presence of a secondary phr mutation in that stock. Since the mus306 and mus308 stocks also carry secondary phr mutations, their partial deficiency in repair of pyrimidine dimers may also be the result of that secondary mutation. Accordingly, the Drosophila mutations that are now definitively associated with defects in the incision step of pyrimidine dimers removal are mei-9, mus201 and phr. The genes mus302 and mus310 appear to play a role in later stages of excision repair.

Repair of double-strand DNA breaks in Drosophila.

This study describes the repair kinetics of DNA double-strand breaks in primary and established cell cultures of Drosophila melanogaster. Double-strand breaks, induced by X-irradiation, were monitored by neutral elution. In primary cell cultures 50% of the double-strand breaks induced by 10 kR of X-rays are repaired within 45 min and 80% of the breaks are repaired within 2-3 h. Repair kinetics in established cell cultures are similar; 50% of the induced breaks are repaired within 20 min and 88% within 3 h. Mutants deficient in other types of DNA repair were also assayed for their capacity to repair double-strand breaks.

Nuclease modification in Chinese hamster cells hypersensitive to DNA cross-linking agents--a model for Fanconi anemia.

Fanconi anemia is a human inherited disease that is characterized by cellular hypersensitivity to DNA cross-linking agents. A number of potential experimental models for that disorder have been developed by selecting mutants that are hypersensitive to bifunctional mutagens. The six mutants of that class in Drosophila, all of which map to the mus308 locus, express an alteration in a mitochondrial nuclease. A recent extension of that observation to cell lines from complementation group A of Fanconi anemia has established a new cellular phenotype for that disorder. In the current study an analogous enzyme has been analyzed in eight recently isolated Chinese hamster cell lines that are hypersensitive to cross-linking agents. Among these lines. V-H4 and V-B7 are shown to exhibit an enzyme modification analogous to that observed in the mutant Drosophila and human cells. These results validate the nuclease assay as an indicator of the Fanconi defect and further establish the V-H4 cell line as a valuable cellular model for analysis of the Fanconi A defect.

X-ray sensitivity and single-strand DNA break repair in mutagen-sensitive mutants of Drosophila melanogaster.

Mutants of Drosophila melanogaster that are sensitive to chemical mutagens were analyzed for sensitivity to X-rays and for the capacity to repair single-strand DNA breaks induced by X-rays. Analysis of X-ray sensitivity demonstrated that 74% of the mutants assayed display some X-ray sensitivity, with 75% of the sensitive lines being extremely sensitive. Repair of single-strand breaks was assayed after both high and low doses of irradiation in order to permit detection of repair over a wide range of damage. The results of this investigation fail to show a correlation between X-ray sensitivity and this particular repair process. Repair of single-strand breaks is therefore mediated by repair processes unrelated to those that are disrupted in the current mutant collection.

A new DNA polymerase species from Drosophila melanogaster: a probable mus308 gene product.

Harris et al. [P.V. Harris, O.M. Mazina, E.A. Leonhardt, R.B. Case, J.B. Boyd, K.C. Burtis, Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes, Mol. Cell. Biol., 16 (1996) 5764-5771.] reported the molecular cloning of Drosophila mus308 gene, and its nucleotide and protein sequences similar to DNA polymerase I. In the present study, we attempted to find and isolate the gene product by purifying a DNA polymerase fraction not present in mus308 flies. A new DNA polymerase with properties different from those of any known polymerase species was identified and partially purified from the wild-type fly embryos through ten column chromatographies. The enzyme was resistant to aphidicolin, but sensitive to ddTTP and NEM. Human proliferating cell nuclear antigen (PCNA) and Drosophila replication protein A (RP-A) did not affect the polymerase activity. It preferred poly(dA)/oligo(dT) as a template-primer. The molecular mass was about 230 kDa with a broad peak region of 200 to 300 kDa in HiPrep16/30 Sephacryl S-300 gel filtration. These properties a different from those of all reported Drosophila polymerase classes such as alpha, beta, gamma, delta, epsilon and zeta and closely resemble those of the gene product expected from the nucleotide sequence. The new polymerase species appears to have ATPase and 3'-5' exonuclease activities as shown by the chromatographies.

Alteration of a nuclease in Fanconi anemia.

Fanconi anemia is a cancer-prone disease characterized by progressive loss of blood cells, skeletal defects and stunted growth. Studies of a nuclease acting on double-stranded DNA have revealed an enzyme alteration in cells derived from Fanconi patients. A particulate fraction isolated from cultured human lymphoblasts and fibroblasts was solubilized with detergent and subjected to isoelectric focusing. Nuclease activity observed in four normal cell lines bands in a pH gradient with a pI of 6.3. Four cell lines belonging to complementation group A exhibit an increase in the pI of that nuclease to 6.8. These observations provide a new diagnostic for this disorder. Analysis of this enzyme in tetraploid cultures derived from fusion of normal and Fanconi cells suggest that the normal phenotype is dominant. That observation supports the hypothesis that the Fanconi A gene is required for modification of the nuclease pI. Definition of the molecular basis of this enzyme alteration should provide insight into the primary genetic lesion in this disorder.

Heat-induced tenderisation of meat by endogenous carboxyl proteases.

The rôle of carboxyl proteases in tenderising meat was investigated by injecting the inhibitors, pepstatin and EPNP, into pre-rigor muscle. The increase in shear force values induced by these inhibitors provided a minimum estimate of the extent to which endogenous carboxyl proteases normally tenderise meat at 60°C. Gel electrophoresis showed that connectin was hydrolysed to a greater extent than other muscle proteins at this temperature and that breakdown of connectin was inhibited by pepstatin and EPNP. Thus it is likely that, when intact, connectin may contribute to the strength of cooked meat.

The effect of temperature and ultimate pH on the increase in meat toughness resulting from restraint during cooking.

Samples of stretched muscle cooked at 50, 60, 70 or 80°C, while restrained at either their original pre-cooking length or further tensioned at about 130% of their original pre-cooking length, had significantly (P < 0·001) greater Warner-Bratzler (WB) peak shear force values for all temperatures than similar samples cooked without restraint except for those restrained at their original length and cooked at 50°C. Restraint during cooking at 80°C increased the peak shear force values of stretched sheep muscles with ultimate pH values in the range 5·5-7·0. This increase, which has been related to connective tissue strength, was not significantly related to ultimate pH. Both initial yield and peak force values, for samples cooked either restrained or unrestrained, decreased significantly (P < 0·001) and at similar (not significantly different) rates with increase in ultimate pH.

Influence of pH on the Warner-Bratzler shear properties of mutton.

Initial yield and peak shear force values obtained for stretched muscles cooked at 80°C for different times decreased linearly at a similar rate with increasing pH, which is consistent with the prime effect of pH being on the myofibrillar structure. The tenderising effect of pressure-heat treatment on stretched and cold-shortened muscle decreased rapidly with increase in ultimate pH until, at values near 7, the effect disappeared. Increased ultimate pH effectively eliminated the large increase in shear force values, occurring in cold-shortened muscle of normal pH and attributable to heat denaturation of myosin, as cooking temperature was increased above 60°C.

A comparison of the meat properties of pasture-fed steers, heifers, pregnant heifers and spayed heifers.

Mean ultimate pH values, sarcomere lengths, cooking losses, Warner-Bratzler peak shear values and compression values for the M. longissimus dorsi (LD) did not differ significantly between heifers, spayed heifers, heifers fitted with a HEIGRO(TM) device(∗) pregnant heifers (2 to 6 months pregnant) and steers, killed at 18 or 22 months of age, with mean carcass weights of 189 and 221 kg, respectively. This result, from animals grazed at pasture, agrees with those of others who used LD samples from lot-fed animals. It would appear that carcasses of non-pregnant heifers, spayed heifers, pregnant heifers, heifers fitted with a HIEGRO(TM) device and steers of similar age and subcutaneous fat depth, do not need to be differentiated on grounds of tenderness.

Comparison of some properties of beef from animals homozygous or heterozygous for muscular hypertrophy.

Carcass characteristics and meat properties of fifty-eight steers and heifers heterozygous (M +) for muscular hypertrophy were compared with those of fifteen homozygous (M M) steers and heifers. The M M animals had a greater carcass weight (173 versus 162 kg) and killing-out percentage than M+ animals. Age at slaughter (298 days for M M and 304 for M +) and liveweights (280 kg, M M, versus 287 kg) did not differ significantly. Carcasses from M M animals were significantly shorter, with a greater eye-muscle area and thinner subcutaneous fat cover over the loin, than those from M + animals. M. semitendinosus (ST) muscles had similar sarcomere lengths, ultimate pH values and weight losses during cooking-80°C for 90 min-for M M and M + animals. Adhesion values, both peak force and work done, were less for ST samples from M M than M + animals, indicating that the ST of M M animals was potentially more tender. This, and other work, suggests that the mechanical properties of cooked ST samples of M+ animals are intermediate between those of M M and + + animals with the ST of M M animals being the most tender. No dark-cutting, high ultimate pH (>6·0) samples were found in the fifteen M M animals slaughtered commercially in the present experiment.

Electrical stimulation of beef sides.

Sides of beef were stimulated with one of three different systems of stimulation-a high voltage system (1100 V), a low voltage system (110 V) and an extra low voltage system (45 V). Warner-Bratzler shear measurements indicated that for muscles removed at 1 or 2 h post slaughter and then subjected to conditions which induce cold shortening, the high voltage system was superior to the other two. Warner-Bratzler shear values and taste panel tenderness scores indicated that, for all stimulation treatments, muscles removed from stimulated sides at 22 h post slaughter were more tender than those removed from control sides at the same time. Muscles from sides which had received the high voltage treatment, but not those from sides which had received the other stimulation treatments, were judged by the taste panel to be less juicy than muscles from the control sides. Lower shear values for individual muscles from stimulated sides were usually accompanied by longer sarcomeres and it is suggested that the major effect of electrical stimulation is the prevention of cold shortening.

Comparison of some properties of meat from normal steers and steers heterozygous for muscular hypertrophy.

Carcase characteristics and meat properties of 19 normal steers and 14 steers heterozygous for muscular hypertrophy (HMH), killed at a liveweight of circa 360 kg and 500 days of age, were compared. Carcases of the HMH animals had less subcutaneous fat and a greater eye muscle area. It was considered that HMH animals were no more stress susceptible than control animals as plasma glucose concentrations at slaughter and ultimate pH values of the M. semitendinosus (ST) did not differ. ST sarcomere length and cooking losses did not differ between control and HMH animals. ST Warner-Bratzler (peak) shear force values and adhesion values were less in the HMH animals; these lower values would indicate that the ST of the HMH animals was more tender. These differences may have resulted from the difference in the mean age of the normal (527 days) and the HMH groups (489 days) at slaughter. However, other workers have found a reduced hydroxyproline content in muscles of homozygous double-muscled animals and the lower adhesion values of animals heterozygous for muscular hypertrophy may reflect the fact that they also have a lower connective tissue content in their muscles compared with normal animals.

Effect of pressure treatments on the mechanical properties of pre- and post-rigor meat.

Pressure-heat treatment of beef semitendinosus samples post-rigor gave shear and tensile results similar to those obtained with pressure treatment pre-rigor. Post-rigor pressure-heat treatment did not affect the contraction state, unlike pre-rigor pressure treatment which caused samples to contract by about 40%. Maximum tenderizing effect by pressure-heat treatment (150 M Nm(-2) at 60°C for 30 min) was achieved when samples were heated at 45°C for 45-180 min immediately before application of the treatment. As the pre-pressurization temperature was increased, the duration of heating became more critical until at temperatures ≥ 60°C the effects of subsequent pressure-heat treatment became very small. Pressure-heat treated samples did not show the increase in shear force values for cooking temperatures ≥ 60°C associated with myofibrillar hardening. It was concluded that pressure-heat treatment primarily affected the myofibrillar structure.