Divergent effects of invasive macrophytes on population dynamics of a snail intermediate host of Schistosoma Mansoni.

Vectors and intermediate hosts of globally impactful human parasites are sensitive to changes in the ecological communities in which they are embedded. Sites of endemic transmission of human schistosome can also be invaded by nonnative species, especially aquatic plants (macrophytes). We tested the effects on macrophyte invasions on experiment snail and schistosome populations created in 100 L mesocosm tanks. We established macrophyte-free mesocosms and those containing one of four widespread macrophyte species that are inedible to snails (duckweed, hornwort, water lettuce, or water hyacinth) and then tracked edible resources (periphyton algae) and the abundance, reproduction, and infection of snail intermediate hosts for 16 weeks. We predicted that the three floating macrophytes would reduce periphyton, thereby reducing snail reproduction, abundance, and infections. In contrast, we predicted that hornwort, which is submerged and provides substrate for periphyton growth, would increase snail reproduction and abundance. As predicted, all floating macrophytes decreased periphyton, but only water hyacinth significantly decreased snail reproduction and abundance. Snail abundance increased significantly only with water lettuce. We hypothesize that this unanticipated increase in snails occurred because water lettuce produced abundant and/or high quality detritus, subsidizing snails despite low periphyton availability. Unfortunately, we detected too few infections to analyze. Aquatic macrophytes exert strong species-specific effects on snail populations. Therefore, efforts to manage invasive plants in endemic sites should evaluate changes in resources, snails, and transmission potential. We recommend caution with management efforts that produce large amounts of detritus, which might stimulate snail populations and therefore risk of human exposure.

Effect of post-brushing mouthwash solutions on salivary fluoride retention--study 2.

OBJECTIVE: This study evaluated the effects of three post-brushing mouthwashes containing 0 ppm F, 225 ppm F, and 500 ppm F, respectively, on salivary fluoride retention after brushing with 1450 ppm fluoride (as NaF) toothpaste and rinsing with water immediately after brushing. METHODS: In this three-phase, randomized, cross-over study, an ion-specific electrode was used to measure salivary F levels in thirty trial participants before brushing (Time 0), and after brushing, rinsing with water, and then rinsing with one of the three mouthwashes. Time points evaluated after brushing were one, three, five, 10, 20, 30, 45, and 60 minutes. For saliva sample collections, subjects were asked to pool saliva in their mouths for 10 seconds before spitting out into a container for each of the time points. RESULTS: The AUC0-60 means for F in saliva were 554, 252, and 20 for the 500, 225, and 0 ppm F mouthwash groups, respectively. The 500 ppm F mouthwash resulted in a 2660% increase in total fluoride salivary retention over 60 minutes when compared with the 0 ppm F group, and a 120% increase when compared with the 225 ppm F group. A significant difference (p < 0.001) in the AUC0-60 means between the three groups was observed using analysis of variance (ANOVA). Paired t-tests also showed significant differences in the mean fluoride retention over 60 minutes for all three pair-wise group comparisons (p < 0.001). CONCLUSION: Use of a fluoride mouthwash containing 225 ppm F or 500 ppm F produced a significant increase in salivary fluoride retention following brushing with a 1450 ppm F toothpaste and rinsing with water compared to rinsing without fluoride. The use of the 500 ppm F mouthwash may be of particular benefit to those at high caries risk.

Study of temperature-growth interactions of entomopathogenic fungi with potential for control of Varroa destructor (Acari: Mesostigmata) using a nonlinear model of poikilotherm development.

AIMS: To investigate the thermal biology of entomopathogenic fungi being examined as potential microbial control agents of Varroa destructor, an ectoparasite of the European honey bee Apis mellifera. METHODS AND RESULTS: Colony extension rates were measured at three temperatures (20, 30 and 35 degrees C) for 41 isolates of entomopathogenic fungi. All of the isolates grew at 20 and 30 degrees C but only 11 isolates grew at 35 degrees C. Twenty-two isolates were then selected on the basis of appreciable growth at 30-35 degrees C (the temperature range found within honey bee colonies) and/or infectivity to V. destructor, and their colony extension rates were measured at 10 temperatures (12.5-35 degrees C). This data were then fitted to Schoolfield et al. [J Theor Biol (1981)88:719-731] re-formulation of the Sharpe and DeMichele [J Theor Biol (1977)64:649-670] model of poikilotherm development. Overall, this model accounted for 87.6-93.9% of the data variance. Eleven isolates exhibited growth above 35 degrees C. The optimum temperatures for extension rate ranged from 22.9 to 31.2 degrees C. Only three isolates exhibited temperature optima above 30 degrees C. The super-optimum temperatures (temperature above the optimum at which the colony extension rate was 10% of the maximum rate) ranged from 31.9 to 43.2 degrees C. CONCLUSIONS: The thermal requirements of the isolates examined against V. destructor are well matched to the temperatures in the broodless areas of honey bee colonies, and a proportion of isolates, should also be able to function within drone brood areas. SIGNIFICANCE AND IMPACT OF THE STUDY: Potential exists for the control of V. destructor with entomopathogenic fungi in honey bee colonies. The methods employed in this study could be utilized in the selection of isolates for microbial control prior to screening for infectivity and could help in predicting the activity of a fungal control agent of V. destructor under fluctuating temperature conditions.

The intracytoplasmic domain of the Env transmembrane protein is a locus for attenuation of simian immunodeficiency virus SIVmac in rhesus macaques.

The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat, rev, and nef are not affected by these changes. In this study, four juvenile macaques received SIVmac-M4 by intravenous injection. Plasma viremia, as measured by branched-DNA (bDNA) assay, reached a peak at 2 weeks postinoculation but dropped to below detectable levels by 12 weeks. At over 1.5 years postinoculation, all four juvenile macaques remain healthy and asymptomatic. In a subsequent experiment, four neonatal rhesus macaques were given SIVmac-M4 intravenously. These animals exhibited high levels of viremia in the acute phase (2 weeks postinoculation) but are showing a relatively low viral load in the chronic phase of infection, with no clinical signs of disease for 1 year. These findings demonstrated that the intracytoplasmic domain of the transmembrane Env (Env-TM) is a locus for attenuation in rhesus macaques.

Pathogenic conversion of live attenuated simian immunodeficiency virus vaccines is associated with expression of truncated Nef.

Rhesus macaques infected with simian immunodeficiency virus (SIV) containing either a large nef deletion (SIVmac239Delta(152)nef) or interleukin-2 in place of nef developed high virus loads and progressed to simian AIDS. Viruses recovered from both juvenile and neonatal macaques with disease produced a novel truncated Nef protein, tNef. Viruses recovered from juvenile macaques infected with serially passaged virus expressing tNef exhibited a pathogenic phenotype. These findings demonstrated strong selective pressure to restore expression of a truncated Nef protein, and this reversion was linked to increased pathogenic potential in live attenuated SIV vaccines.

Fatal immunopathogenesis by SIV/HIV-1 (SHIV) containing a variant form of the HIV-1SF33 env gene in juvenile and newborn rhesus macaques.

SIV/HIV-1 (SHIV) chimeric clones, constructed by substituting portions of the pathogenic molecular clone SIVmac239 with counterpart portions from HIV-1 clones, provide a means to analyze functions of selected HIV-1 genes in vivo in nonhuman primates. Our studies focused on SHIVSF33, which contains the vpu, tat, rev, and env genes of the cytopathic, T-cell line tropic clone HIV-1sf33 (subtype-B); this clone has a premature stop codon in the vpu gene. In three juvenile macaques inoculated intravenously with SHIVSF33, low-level persistent infection was established; no disease was observed for a period of >2 years. However, at approximately 16 months p.i., one of four SHIVSF33-infected juvenile macaques exhibited an increase in virus load, depletion of CD4(+) T cells in peripheral blood and lymph nodes, and other symptoms of simian AIDS (SAIDS). Virus recovered from this animal in the symptomatic stage was designated SHIVSF33a (A, adapted); this virus displayed multiple amino acid sequence changes throughout the HIV-1 env gene compared with the input SHIVSF33 clone. Additionally, a mutation in all clones from SHIVSF33a restored the open reading frame for the vpu gene. In vitro evaluations in tissue-culture systems revealed that SHIVSF33a replicated to higher levels and exhibited greater cytopathicity than SHIVSF33. Furthermore cloned env genes for SHIVSF33a were more fusogenic in a cell-fusion assay compared with the env gene of the SHIVSF33. Intravenous inoculation of SHIVsf33a into juvenile and newborn macaques resulted in a rapid decline in CD4(+) T cells to very low levels and development of a fatal AIDS-like disease. A cell-free preparation of this pathogenic chimeric virus also established persistent infection when applied to oral mucosal membranes of juvenile macaques and produced a fatal AIDS-like disease. These studies on pathogenic SHIVSF33a establish the basis for further investigations on the role of the HIV-1 env gene in virus adaptation and in mechanism(s) of immunodeficiency in primates; moreover, the chimeric virus SHIVSF33a can play a role in elucidating mucosal membrane transmission and development of antiviral vaccines in newborns as well as juvenile and adult macaques.

Importance of the intracytoplasmic domain of the simian immunodeficiency virus (SIV) envelope glycoprotein for pathogenesis.

SIVmac1A11 and SIVmac239 are nonpathogenic and pathogenic molecular clones in rhesus macaques, respectively. Although these viruses exhibit approximately 98% nucleotide and amino acid sequence homology, differences are found in the length of the translation frames for several genes. SIVmac239 has a premature stop codon in nef, whereas SIVmac1A11 has a premature stop codon in vpr and two premature stop codons in the intracytoplasmic domain of the env-transmembrane (TM) subunit. Recombinant viruses, constructed through reciprocal exchange of large DNA restriction enzyme fragments between SIVmac1A11 and SIVmac239, were evaluated in adult rhesus macaques. This in vivo analysis revealed that two or more regions of the SIVmac genome were essential for high virus load and disease progression (Marthas et al., 1993. J. Virol. 67, 6047-6055). An important gap in knowledge remaining from this study was whether the premature stop codons in env-TM of recombinant virus SIV1A11/239gag-env/1A11 (Full-length vpr and nef, two stop codons in env-TM) reverted to coding triplets in vivo. Here, we report that viral sequences in macaques, which succumbed to an AIDS-like disease after infection with SIV1A11/239gag-env/1A11, exhibited reversion of both env-TM stop codons. In addition, antibodies to the intracytoplasmic domain of env-TM were detected in macaques containing revertant virus and showing disease; this finding indicates that this domain of the env glycoprotein was expressed in vivo. Thus selection for viral variants with full-length env-TM demonstrated that the cytoplasmic domain of the SIVmac env glycoprotein plays a role in viral persistence and immunodeficiency in primates.

Persistent infection of rhesus macaques with T-cell-line-tropic and macrophage-tropic clones of simian/human immunodeficiency viruses (SHIV).

To elucidate the functions of human immunodeficiency virus type 1 (HIV-1) genes in a nonhuman primate model, we have constructed infectious recombinant viruses (chimeras) between the pathogenic molecular clone of simian immunodeficiency virus (SIV) SIVmac239 and molecular clones of HIV-1 that differ in phenotypic properties controlled by the env gene. HIV-1SF33 is a T-cell-line-tropic virus which induces syncytia, and HIV-1SF162 is a macrophage-tropic virus that does not induce syncytia. A DNA fragment encoding tat, rev, and env (gp160) of SIVmac239 has been replaced with the counterpart genetic region of HIV-1SF33 and HIV-1SF162 to derive chimeric recombinant simian/human immunodeficiency virus (SHIV) strains SHIVSF33 and SHIVSF162, respectively. In the acute infection stage, macaques inoculated with SHIVSF33 had levels of viremia similar to macaques infected with SIVmac239, whereas virus loads were 1/10th to 1/100th those in macaques infected with SHIVSF162. Of note is the relatively small amount of virus detected in lymph nodes of SHIVSF162-infected macaques. In the chronic infection stage, macaques infected with SHIVSF33 also showed higher virus loads than macaques infected with SHIVSF162. Virus persists for over 1 year, as demonstrated by PCR for amplification of viral DNA in all animals and by virus isolation in some animals. Antiviral antibodies, including antibodies to the HIV-1 env glycoprotein (gp160), were detected; titers of antiviral antibodies were higher in macaques infected with SHIVSF33 than in macaques infected with SHIVSF162. Although virus has persisted for over 1 year after inoculation, these animals have remained healthy with no signs of immunodeficiency. These findings demonstrate the utility of the SHIV/macaque model for analyzing HIV-1 env gene functions and for evaluating vaccines based on HIV-1 env antigens.

Genetic and biological comparisons of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus (SIVmac).

Simian immunodeficiency virus (SIV) is a designation for a group of related but unique lentiviruses identified in several primate species. A viral isolate from a rhesus macaque (i.e., SIVmac) causes a fatal AIDS-like disease in experimentally infected macaques, and several infectious molecular clones of this virus have been characterized. This report presents the complete nucleotide sequence of molecularly cloned SIVmac1A11, and comparisons are made with the sequence of molecularly cloned SIVmac239. SIVmac1A11 has delayed replication kinetics in lymphoid cells but replicates as well as uncloned SIVmac in macrophage cultures. Macaques infected with virus from the SIVmac1A11 clone develop antiviral antibodies, but virus does not persist in peripheral blood mononuclear cells and no disease signs are observed. SIVmac239 infects lymphoid cells, shows restricted replication in cultured macrophages, and establishes a persistent infection in animals that leads to a fatal AIDS-like disease. Both viruses are about 98% homologous at the nucleotide sequence level. In SIVmac1A11, the vpr gene as well as the transmembrane domain of env are prematurely truncated, whereas the nef gene of SIVmac239 is prematurely truncated. Sequence differences are also noted in variable region 1 (V1) in the surface domain of the env gene. The potential implications of these and other sequence differences are discussed with respect to the phenotypes of both viruses. This animal model is critically important for investigating the roles of specific viral genes in viral/host interactions that cannot be studied in individuals infected with the human immunodeficiency virus (HIV).

cis-acting regulatory regions in the long terminal repeat of simian foamy virus type 1.

Simian foamy virus type 1 (SFV-1), a member of the Spumavirinae subfamily of retroviruses, encodes a transcriptional transactivator (taf) that strongly augments gene expression directed by the viral long terminal repeat (LTR) (A. Mergia, K. E. S. Shaw, E. Pratt-Lowe, P. A. Barry, and P. A. Luciw, J. Virol. 65:2903-2909, 1991). This report describes cis-acting regulatory elements in the LTR that control viral gene expression. A series of LTR mutants and hybrid promoter constructs have been analyzed in transient expression assays for responsiveness to Taf. The targets for transactivation have been mapped to two regions of the U3 domain of the LTR, between positions -1196 and -880 and between positions -403 and -125 (+1 represents the transcription initiation site). No significant nucleotide sequence homology between these two regions is noted; thus, the SFV-1 taf gene acts through at least two distinct sequence elements in the LTR. The target contained between positions -403 and -125 acts independently of orientation, in different cell types and species, and in the context of a heterologous promoter. Thus, the target element between positions -403 and -125 has properties of a transcriptional enhancer. The observation that two distinct elements in the SFV-1 LTR are targets for transcriptional transactivation is novel with respect to observations for other retroviral systems. The R-U5 region of the SFV-1 LTR down-regulates transactivation by severalfold. Computer analysis of the R-U5 region revealed a secondary structure with a free-energy level of -74 kcal (ca. -310,000 J); this structural feature may account for the inhibitory effect on gene expression directed by the LTR. Taf of SFV-1 had no effect on gene expression directed by the LTR of the related human foamy virus, whereas Taf transactivates gene expression directed by the LTRs of the human and simian immunodeficiency viruses. Comparative functional analysis of Taf on homologous and heterologous LTRs may facilitate elucidation of the mechanism of transactivation of foamy viruses.

Identification of the simian foamy virus transcriptional transactivator gene (taf).

Simian foamy virus type 1 (SFV-1), a member of spumavirus subfamily of retroviruses, encodes a transcriptional transactivator that functions to strongly augment gene expression directed by the viral long terminal repeat (LTR). The objective of this study was to identify the viral gene responsible for transactivation. Nucleotide sequences between the env gene and the LTR of SFV-1 were determined. The predicted amino acid sequence revealed two large open reading frames (ORFs), designated ORF-1 (311 amino acids) and ORF-2 (422 amino acids). In the corresponding region of the human foamy virus, three ORFs (bel-1, bel-2, and bel-3) have been identified (R. M. Flugel, A. Rethwilm, B. Maurer, and G. Darai, EMBO J. 6:2077-2084, 1987). Pairwise comparisons of the ORF-1 and ORF-2 with bel-1 and bel-2 show small clusters of homology; less than 39% overall homology of conserved amino acids is observed. A counterpart for human foamy virus bel-3 is not present in the SFV-1 sequence. Three species of viral RNA have been identified in cells infected with SFV-1; an 11.5-kb RNA representing full-length transcripts, a 6.5-kb RNA representing the env message, and a 2.8-kb RNA from the ORF region. Analysis of a cDNA clone encoding the ORF region of SFV-1 reveals that the 2.8-kb message is generated by complex splicing events involving the 3' end of the env gene. In transient expression assays in cell lines representing several species. ORF-1 was shown to be necessary and sufficient for transactivating viral gene expression directed by the SFV-1 LTR. The target for transactivation is located in the U3 domain of the LTR, upstream from position - 125 (+ 1 represents the transcription initiation site). We propose that OFF-1 of SFV-1 be designated the transcriptional transactivator of foamy virus (taf).

The "numb cheek-limp lower lid" syndrome.

A patient developed isolated numbness, 1st confined to the lateral nose and upper lip, but later involving the cheek, lower lip, upper gingiva, and the palate. This numbness was later associated with paresis of the muscles of the upper lip and angle of the mouth and with ipsilateral lower lid droop (the "numb cheek-limp lower lid" syndrome). Squamous cell carcinoma was discovered infiltrating the infraorbital nerve and distal branches of the facial nerve. Cheek numbness associated with lower eyelid or upper lip weakness may herald a neoplasm affecting the infraorbital nerve and distal facial nerve branches.

Simian foamy virus type 1 is a retrovirus which encodes a transcriptional transactivator.

Simian foamy viruses, members of the spumavirus subfamily of retroviruses, are found in a variety of nonhuman primates and, as yet, remain to be characterized with respect to genetic structure and regulation of viral gene expression. The genome of simian foamy virus type 1 (SFV-1), an isolate from rhesus macaques, has been molecularly cloned, and the role of the viral long terminal repeat (LTR) in transcriptional control has been investigated. The SFV-1 LTR is 1,621 base pairs long, and sequence comparisons with human foamy virus revealed a pattern of clustered homology. A cap site in the LTR was identified by analysis of SFV-1 transcripts in infected cells. Transient expression assays in cell lines representing several species and different cell types showed that the SFV-1 LTR has low basal activity in uninfected cells, whereas LTR-directed expression is greatly increased in cells infected with SFV-1. This transactivation is mediated by a mechanism involving increases in steady-state levels of viral transcripts. Thus, the SFV-1 genome encodes a transactivator that functions on the LTR at the transcriptional level.

Relationship of the env genes and the endonuclease domain of the pol genes of simian foamy virus type 1 and human foamy virus.

We have molecularly cloned and sequenced a portion of the simian foamy virus type 1 (SFV-1); open reading frames representing the endonuclease domain of the polymerase (pol) and the envelope (env) genes were identified by comparison with the human foamy virus (HFV). Unlike the HFV genomic organization, the SFV-1 pol gene overlaps the env gene; thus, the open reading frames reported for HFV between pol and env is not present in SFV-1. Comparisons of predicted amino acid sequences of HFV and SFV-1 reveal that the endonuclease domains of the pol genes are about 84% related. The region predicted to encode the SFV-1 extracellular env domain is 569 codons; SFV-1 and HFV have 64% amino acid similarity in this env domain. The predicted hydrophobic transmembrane env proteins of both HFV and SFV-1 show about 73% similarity. A total of 16 potential glycosylation sites are found in SFV-1 env, and 15 are found in HFV; 11 are shared. SFV-1 has 25 cysteine residues, and HFV has 23 residues; all 23 cysteine residues of HFV are conserved in SFV-1. This sequence analysis reveals that the human and simian foamy viruses are highly related.

En bloc cervical lipectomy for treatment of the problem neck in facial rejuvenation surgery.

The treatment of cervical fat in facial aesthetic surgery has received much attention in recent years. Suction lipectomy has become a very popular technique for removing cervical fat because it is easy to perform and results in few complications. This paper describes the en bloc excision of cervical fat in conjunction with rhytidectomy. The senior author has treated 1,000 patients over 17 years using this technique with a high degree of patient satisfaction and minimal morbidity. Although suction lipectomy alone may be indicated for the younger patient, our experience suggests that the en bloc excisional technique is the treatment of choice in the older patient in whom a rhytidectomy is also indicated. In contrast with suction lipectomy, we have found that the en bloc excision of cervical fat allows for more anatomic dissection and facilitates removal of greater amounts of fat and better redraping of the cervical skin.

A new feature of the ablepharon macrostomia syndrome: zygomatic arch absence.

The ablepharon macrostomia syndrome is an extremely rare congenital anomaly. It is characterised by bilateral absence or hypoplasia of lower eyelids, macrostomia and multiple other congenital anomalies. Three cases have been reported (McCarthy and West, 1977; Hornblass and Reifler, 1985). In addition to ablepharon and macrostomia, other anomalies common to all patients include auricular deformity, nasal alar deformity, absence of lanugo hair, dry, ichthyotic skin and ambiguous genitalia. A new feature of the syndrome is described--absence of the zygomatic arches. In addition, an expanded and revised classification of the ablepharon macrostomia syndrome and related disorders is presented. Skin graft pigmentation in this black patient has been prevented by prolonged application of sun block.

Amino-terminal deletion mutants of the Rous sarcoma virus glycoprotein do not block signal peptide cleavage but can block intracellular transport.

Protein sequence requirements for cleavage of the signal peptide from the Rous sarcoma virus glycoprotein have been investigated through the use of deletion mutagenesis. The phenotypes of these mutants have been characterized by expression of the cloned, mutated env genes in CV-1 cells using a late replacement SV40 vector. The deletion mutations were generated by Ba131 digestion at the XhoI site located near the 5' end of the coding sequence for the structural protein gp85, which is found at the amino terminus of the precursor glycoprotein, Pr95. The results of experiments with three mutants (X1, X2, and X3) are presented. Mutant X1 has a 14 amino acid deletion encompassing amino acids 4-17 of gp85, which results in the loss of one potential glycosylation site. In mutants X2 and X3 the amino terminal nine and six amino acids, respectively, of gp85 are deleted. During the biosynthesis of all three mutant polypeptides, the signal peptide is efficiently and accurately cleaved from the nascent protein, even though in mutants X2 and X3 the cleavage site itself has been altered. In these mutants the alanine/aspartic acid cleavage site has been mutated to alanine/asparagine and alanine/glutamine, respectively. These results are consistent with the concept that sequences C-terminal to the signal peptidase site are unimportant in defining the site of cleavage in eucaryotes. Mutants X2 and X3 behave like wild-type with respect to protein glycosylation, palmitic acid addition, cleavage to gp85 and gp37, and expression on the cell surface. Mutant X1, on the other hand, is defective in intracellular transport. Although it is translocated across the rough endoplasmic reticulum and core-glycosylated, its transport appears to be blocked at an early Golgi compartment. No terminal glycosylation of the protein, cleavage of the precursor protein to the mature products, or expression on the cell surface is observed. The deletion in X1 thus appears to destroy signals required for export to the cell surface.

Postreplication repair defects in mutants of Drosophila melanogaster.

Mutants of Drosophila melanogaster which are defective in DNA synthesis have been identified among mutagen-sensitive stocks through analysis of both organ and cell cultures. A new procedure employing larval brain ganglia allows poorly fertile or sterile mutants to be analyzed for the first time. Parallel studies were performed in both tissues to establish the sensitivity of the new assay relative to that of the proven cell-culture assay. Damage was induced in the DNA of cultured cells with UV irradiation and in that of ganglial cells with the carcinogen N-acetoxy-2-acetylaminofluorene. Cultures were then pulse-labeled with 3H-thymidine, incubated in the absence of thymidine, and the newly synthesized DNA was analyzed by alkaline sucrose gradient centrifugation. The molecular weight of labeled DNA from mutant cells was compared with that from control cells to assess the effect of the mutant on DNA synthesis. Among 16 mutant stocks that were scanned in either or both tissues, seven show reductions in DNA synthesis using an undamaged template. Mutants at five different genetic loci [mus(2)205, mus(3)304, mus(3)308, mus(3)310 and mus(3)311] possess a reduced capacity to synthesize DNA on a UV-damaged template in primary cell cultures. Four of these five defects can also be detected in carcinogen-treated organ cultures. Two additional defects in postreplication repair were observed with the brain-ganglia assay in strains that cannot be assayed in cell culture [mus(1)108, mus(2)206].

Genetic analysis of X-linked mutagen-sensitive mutants of Drosophila melanogaster.

Mutants at 2 new loci which control mutagen-sensitivity are described. Mutants of both foci are female-sterile and are hypersensitive to killing by MMS; neither increases the frequency of sex-linked recessive lethals. A screen of previously described female-sterile and meiotic mutants has revealed that a number of these are also sensitive to mutagens. In addition, several new mutants have been identified on the basis of sensitivity to either HN2 or MMS. An analysis of complementation data suggests that all of the X-linked genes controlling sensitivity to MMS may now have been identified. Among the new mei-41 alleles are mutants which show very little meiotic nondisjunction or loss. Cytogenetic mapping of previously known mutants is also described. The mutants mus(1)104D1 and mei-41D5 are located in the region 14B13+/- -14D1,2 on the polytene chromosome map, and they map very close to each other genetically. Cytogenetically mus(1)101D1 is between salivary chromosome bands 12A6,7 and 12D3, mus(1)103D1 is between bands 12A1,2 and 12A6,7 and mus(1)109A1 is in section 8F3--9A2.

Third-chromosome mutagen-sensitive mutants of Drosophila melanogaster.

A total of 34 third chromosomes of Drosophila melanogaster that render homozygous larvae hypersensitive to killing by chemical mutagens have been isolated. Genetic analyses have placed responsible mutations in more than eleven complementation groups. Mutants in three complementation groups are strongly sensitive to methyl methanesulfonate, those in one are sensitive to nitrogen mustard, and mutants in six groups are hypersensitive to both mutagens. Eight of the ten loci mapped fall within 15% of the genetic map that encompasses the centromere of chromosome 3. Mutants from four of the complementation groups are associated with moderate to strong meiotic effects in females. Preliminary biochemical analyses have implicated seven of these loci in DNA metabolism.