Procurement of spore‐free Bacillus anthracis for molecular typing outside of BSL3 environment

Aims: To (i) develop a protocol that would eliminate or greatly reduce sporulation within Bacillus anthracis vegetative cells, and (ii) harvest an adequate number of cells and sufficient DNA suitable for molecular methods including Riboprint^® analysis and pulse field gel electrophoresis (PFGE). Methods and Results: Seven strains of B. anthracis (Ames, French B2, Heluky, Kruger, Pasteur, Sterne, and Vollum) were grown at 37, 42 and 45°C under normal air, enhanced CO₂, microaerophilic, and anaerobic conditions on solid media and subcultured in two broths with and without supplements. The bacterial cells were centrifuged and washed. Slides made from the cell pellets were stained with Malachite Green and observed for the presence of spores. Cell preparations were subjected to 80°C for 30 min and processed for and analysed by either Riboprinter^® or PFGE. Multiple pellets of each strain were processed, stained, placed onto solid culture media, incubated for 7 days and observed for growth. The cell preparations yielded clear and reproducible results with both molecular methods. None of the cell preparations yielded growth on the culture media. Conclusions: This method eliminated viable spores in cell preparations of B. anthracis, yet still allowed the growth of vegetative cells to provide sufficient DNA suitable for analysis by Riboprinter^® and PFGE. Significance and Impact of the Study: This method will provide safe cell preparations, prevent instrument contamination, and may be useful for other aerobic and anaerobic spore‐formers.     

DNA extraction from fixed cytogenetic cell suspensions

We developed a procedure for DNA extraction from small volumes of fixed cell suspensions previously prepared for conventional cytogenetic analysis. Good quality DNA was isolated with a fast and simple protocol using DNAzol reagent. This provided suitable DNA for various types of molecular analyses, including polymerase chain reaction, restriction fragment length polymorphism, denaturing high-performance liquid chromatography, and direct sequencing. This technique provides sufficient material for such test, which are important for diagnosis of neoplastic diseases in pediatric patients.     

Ageing of fixed cytological preparations produces degradation of chromosomal DNA

When fixed chromosome preparations were allowed to age for 1-72 h, they became progressively more susceptible to digestion by exonuclease III and by S1 nuclease. Analysis of DNA from these aged preparations on agarose gels showed that the molecular weight of the DNA decreased as ageing progressed. We conclude that DNA in fixed chromosome preparations becomes progressively degraded as the preparations age.     

Improved confocal characterization of targets in nuclei of cytogenetic preparations

OBJECTIVE: To show that cellular preparations requiring depth analysis of different domains stained by molecular cytogenetic methods (fluorescence in situ hybridization and primed in situ) can be improved by regularized factor analysis of medical image sequences (FAMIS) to isolate fluorescent probes by means of intensity depth profiles of fluorochromes, to track relevant DNA sequences (cosmids and centromeres) in cell nuclei during interphase and to improve the use of cytogenetic techniques resulting in flat preparations of whole cells that are assumed to preserve probe access to their targets. STUDY DESIGN: 3D sequences of images obtained by depth displacement in a confocal microscope were first analyzed by the FAMIS algorithm, which provides factor curves. Factor images then resulted from regularization methods that improve signal/noise ratio while preserving target contours. RESULTS: Factor curves and regularized factor images helped analyze targets inside nuclei. CONCLUSION: It is possible to process preparations containing numerous spots (even when they are on different planes) to differentiate stained targets, to investigate depth differences and to improve visualization and detection.     

Maternal cell contamination of cultures of spontaneous abortion fibroblasts: importance for cytogenetic analysis of embryonic lethality

The results of standard cytogenetic analysis of the long-term cultures of embryonic fibroblasts of 478 first-trimester spontaneous abortions were retrospectively reviewed. In 16% of embryos with cytogenetically confirmed karyotype 46,XX, the Y chromosome was found by molecular genetic methods. Prior to obtaining the chromosome preparations, the cell cultures of Y chromosome-carrying embryos were maintained for a longer period than the cultures of embryos without the Y chromosome. Thus, a late entry of a culture into the logarithmic growth phase serves as marker of maternal cell contamination. We developed a mathematic model for assessment of karyotype incidence and the "sex ratio" of spontaneous abortions, taking into account risk of maternal cell contamination in extraembryonic tissue cultures. Thus estimated, the incidence of chromosomal abnormalities in the studied sample increased from 54.6 to 60.3% and the expected sex ratio increased from 0.66 to 1.02 in abortions with normal karyotype. Using molecular analysis of inheritance of polymorphic DNA markers of six autosomes (2, 11, 16, 19, 20, and 21), the proposed model was tested on 60 embryos with karyotype 46,XX and their parents. Numerical chromosome abnormalities were revealed in uncultured tissues of seven abortions (11.7%), including four without the Y chromosome, which is in a good agreement with the expected incidence of karyotype abnormalities (8.3%) predicted by our model. In view of this, estimating risk of maternal cell contamination in embryonic cell cultures seems necessary for correctly assessing the effect of natural selection in humans, for understanding the mechanisms that determine the sex ratio, and for evaluating the precision of prenatal cytogenetic diagnosis of chromosomal abnormalities.     

Cytogenetic analysis of chorionic villi: a technical assessment

Summary Eighty-five samples of chorionic villi from women undergoing prenatal diagnosis at 8 to 12 weeks' gestation were subjected to cytogenetic analysis. Samples were prepared by a direct technique that permits limited analysis within two hours and by a short-term culture technique that permits detailed structural analysis within one week. An adequate number of cell divisions for cytogenetic analysis was obtained from 96% of living fetuses. Using both the direct technique and short-term culture, satisfactory banded chromosomal preparations were made in 93% of cases. Eleven of 12 pregnancies (92%) shown by ultrasound to be dead shortly before sampling, had cytogenetic abnormalities. Further studies are needed to develop banding definition equivalent to that available on cultured amniocytes.     

Detection and chromosomal assignment of SV40-DNA integration in Chinese hamster cell lines by chromosome sorting and dot blot hybridization

A combination of cytometric (chromosome sorting), molecular (dot blot hybridization using radio-active and/or biotinylated DNA probes) and cytogenetic (G-banding) evaluation is described which allows the rapid identification of single copy and repetitive viral integrates and their assignment to chromosome groups or even individual chromosomes. In the case of Chinese hamster cell line CO 631 it could be demonstrated that SV40 DNA was solely integrated into a submetacentric marker chromosome. Such a cytometric/molecular/cytogenetic "identogram" may prove to be a useful tool in many areas of cell and tumor biology. Furthermore, amounts of chromosomes sufficient for analysis as well as subsequent cloning experiments can be accumulated.     

A new procedure for the simultaneous large-scale purification of bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase

A procedure for simultaneous large-scale purification of the bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase has been developed. The method involves bacterial cell disruption by sonication, fractionation of cell extract with polymin P, salt elution from the polymin pellets, ammonium sulfate precipitation, and subsequent column chromatography purification of the enzymes. To enrich the enzyme content highly in the initial source non-permissive Escherichia coli B-23 cells infected with T4 amN82 phage were used. The procedure described is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 200 g cell paste. It can be easily scaled up. The method results in large amounts of the enzymes with very high specific activities, good stability essential lacking exonuclease and endonuclease contamination. The final enzyme preparations were efficiently used in DNA sequencing and in multiple experiments on construction of various recombinant DNAs for cloning and expression in vivo.     

Molecular and Immunohistochemical Characterization of Historical Long-Term Preserved Fixed Tissues from Different Human Organs

University and museum collections are very important sources of biological samples that can be used to asses the past and present genetic diversity of many species. Modern genetic and immunohistochemical techniques can be used on long-term preserved fixed tissues from museum specimens to answer epidemiological questions. A proof of principle was established to apply modern molecular genetics and immunohistochemical methods to these old specimens and to verify the original diagnosis. We analysed 19 specimens from our university collection including human organs that had been in fixative for more than 80 years. The tissues originated from lung, colon, brain, heart, adrenal gland, uterus and skin. We isolated amplifiable DNA from these wet preparations and performed mutational analysis of BRAF, KRAS and EGFR. The tissues were also embedded in paraffin and used for modern histology and immunohistochemistry. Our data show that amplifiable DNA is extractable and ranged from 0.25 to 22.77 μg of total DNA. In three specimens BRAF^V600E or KRAS^G12D mutations were found. Additionally, expression of different proteins like vimentin and GFAP was detected immunohistochemical in six investigated specimens. On the basis of our results the original diagnosis was altered in three specimens. Our work showed that it is possible to extract amplifiable DNA suitable for sequence analysis from long-term fixed tissue. Furthermore, histology and immunohistochemistry is feasible in specimens fixed long time ago. We conclude that these old preparations are suitable for further epidemiological research and that our methods open up new opportunities for future studies.     

Extraction of DNA from exfoliative cytology specimens and its suitability for analysis by the polymerase chain reaction

The extraction of DNA from archival exfoliative cytology samples would allow the molecular biological analysis of this readily available material using the polymerase chain reaction (PCR). We have quantitatively and qualitatively studied the extraction of DNA from a variety of cytological preparations. For both fresh and archival cervical smears, overnight incubation with proteinase K produces high yields of high molecular weight DNA, but simply boiling the samples produces DNA suitable for PCR amplification of a single copy gene. Increasing the proteinase K incubation to several days allows the extraction of DNA from fixed and stained archival cytology slides from a variety of sites. The extracted DNA was again suitable for PCR analysis. Fresh and archival cytological material can be utilized for molecular biological study of disease processes using PCR. Archival cytological material is probably the best source of DNA and RNA after stored frozen tissue.     

Chromosomal abnormalities and DNA image cytometry of haematological neoplasms in fine needle aspirates of lymph nodes

The current diagnostics of haematological neoplasms along with morphological analysis, immunophenotyping and molecular analysis inevitably includes cytogenetic analysis. In this work the possibility of cytomorphological subclassification of haematological neoplasms from lymph node fine needle aspirates was examined without depending upon the referential histological diagnosis and cytogenetic analysis. In addition, the feasibility of cytogenetic analysis of the material obtained by lymph node fine needle aspiration (FNA) was examined. By analysing the findings of cytogenetic analysis and DNA image cytometry, it was decided to examine the possibility of comparing the findings and supplementing diagnostic possibilities of these methods. In 15 cases cytological diagnoses and cytogenetic analysis of haematological neoplasms were performed on the material obtained by lymph node FNA. In 12 of 15 cases histological diagnosis was made separately. A good cytohistological correlation was available in 9 of 12 cases (75%). Cytomorphological diagnoses in 10 of 15 cases (76%) were confirmed by the finding of a specific chromosomal translocation. In two cases cytological diagnosis did not correlate with the histological diagnosis and was confirmed only with specific chromosomal translocations. The lymphocytes obtained by lymph node FNA were adequate material for cytogenetic analysis - in 15 of 18 (83%) cases mitoses in cell cultures were obtained. In 13 of 15 (87%) cases clonal chromosomal abnormalities were detected, whereas in 2 of 15 (13%) cases a normal karyotype was found. DNA image cytometry was performed on nine samples, whereas in six samples the material was not sufficient. Although a small number of samples was analysed in the cases with identical cytomorphological diagnoses, the analysed histograms regarding the DNA index values showed heterogeneity. In conclusion, a cell culture sampled by FNA of lymph nodes is an adequate method for the chromosomal analysis. The specific cytogenetic abnormality associated with cytological diagnosis provides an opportunity to make a definitive diagnosis and provides a powerful approach when reference diagnosis on biopsy material cannot be obtained.     

INCORPORATION OF NUCLEOTIDES INTO NUCLEI OF FIXED CELLS BY DNA POLYMERASE

The enzyme calf thymus polymerase requires denatured or single-stranded DNA as a primer for DNA synthesis and is inactive on native DNA preparations. The enzyme and tritium-labeled deoxyribonucleoside triphosphates were incubated with alcohol-fixed and Carnoy-fixed tissue preparations to see if primer DNA could be found in several types of cells undergoing DNA synthesis. In all cases, low-pH controls were prepared for comparison. Priming activity was not found in nuclei that had been fixed in alcohol. Priming activity was found in cell nuclei that had been fixed with an acid fixative or had been treated at a low pH prior to treatment with the enzyme reaction mixture.     

Maternal Cell Contamination of Cultures of Spontaneous Abortion Fibroblasts: Importance for Cytogenetic Analysis of Embryonic Lethality

The results of standard cytogenetic analysis of the long-term cultures of embryonic fibroblasts of 478 first-trimester spontaneous abortions were retrospectively reviewed. In 16% of embryos with cytogenetically confirmed karyotype 46,XX, the Y chromosome was found by molecular genetic methods. Prior to obtaining the chromosome preparations, the cell cultures of Y chromosome-carrying embryos were maintained for a longer period than the cultures of embryos without the Y chromosome. Thus, a late entry of a culture into the log-growth phase serves as marker of maternal cell contamination. We developed a mathematical model for assessment of karyotype incidence and the sex ratio of spontaneous abortions, taking into account risk of maternal cell contamination in extraembryonic tissue cultures. Thus estimated, the incidence of chromosomal abnormalities in the studied sample increased from 54.6 to 60.3% and the expected sex ratio increased from 0.66 to 1.02 in abortions with normal karyotype. Using molecular analysis of inheritance of polymorphic DNA markers of six autosomes (2, 11, 16, 19, 20, and 21), the proposed model was tested on 60 embryos with karyotype 46,XX and their parents. Numerical chromosome abnormalities were revealed in uncultured tissues of seven abortions (11.7%), including four without the Y chromosome, which is in a good agreement with the expected incidence of karyotype abnormalities (8.3%) predicted by our model. In view of this, estimating risk of maternal cell contamination in embryonic cell cultures seems necessary for correctly assessing the effect of natural selection in humans, for understanding the mechanisms that determine the sex ratio, and for evaluating the accuracy of prenatal cytogenetic diagnosis of chromosomal abnormalities.     

Exfoliative cytologic evaluation of primary cultured lung carcinomas

This paper describes a tissue culture and exfoliative cell culture system that enables one to (1) evaluate the adequacy of primary lung carcinoma cultures for cytogenetic analysis, and (2) predict the likelihood of viable cells and type of differentiation present in the primary lung tumor cultures used for cytogenetics. Primary lung carcinomas were established from explant outgrowths and maintained in serum supplemented or serum free media on plastic or basement membrane associated protein coated dishes in order to obtain cells for karyotypic analysis (Miura et al., 1990). The media from these cultures that would ordinarily have been discarded was aspirated at each media change and used to prepare cytocentrifuge cytology preparations. Papanicolaou stained cells from the preparations were evaluated by cytotechnologists in order to assess (1) the cellularity and presence of cancer cells in the sample, (2) differentiation of the malignant cells, and (3) adequacy for chromosomal studies. We determined that cytology preparations of cell and explant outgrowth cultures from primary lung tumors are a reliable method for screening and evaluating the suitability of primary lung carcinoma cultures for cytogenetic analysis.Supported in part by an NCI grant CA-45745 (JRT). JRT is a Scholar of the Leukemia Society of America.     

Mouse chromosome-specific painting probes generated from microdissected chromosomes

Using degenerate primer amplification of chromosomes microdissected from banded cytogenetic preparations, we constructed both whole chromosome painting probes for mouse Chromosomes (Chrs) 1, 2, 3, and 11 and a centromere probe that strongly paints most mouse centromeres. We also amplified a Robertsonian translocation chromosome microdissected from unstained preparations to construct a painting probe for Chrs 9 and 19. The chromosomes probes uniformly painted the respective chromosomes of origin. We demonstrated the utility of the Chr 11 probe in aberration analysis by staining mutants that we had previously identified as containing a Chr 11 translocation, and in some mutant cell lines we observed chromosome rearrangements not previously detected in stained cytogenetic preparations. The technology of microdissection and amplification applies to all mouse chromosomes or to specific subchromosomal regions and will be useful in mouse genetics, in aberration analysis, and for chromosome identification.     

Glucocorticoid receptor binding to calf thymus DNA. 1. Identification and characterization of a macromolecular factor involved in receptor-steroid complex binding to DNA.

Activation of receptor-steroid complexes to a form with high affinity for DNA is a poorly understood process involving multiple components in addition to the holoreceptor. Employing rat HTC cells as the source of glucocorticoid receptor, we show that maximal receptor binding to calf thymus DNA is mediated by a previously unknown small molecular weight factor. This factor can be removed from cytosolic preparations of receptor by gel filtration chromatography. Salt extraction of crude nuclear pellets afforded much larger amounts of a similar DNA-binding activity factor. The cytoplasmic factor and the more abundant nuclear factor were identical on the basis of their similar physical properties. The factor was precipitable in the crude state with (NH4)2SO4 and stable to heat as well as freezing and thawing. Chromatography on DNA-cellulose revealed that the factor itself did not bind to DNA. The factor could be filtered through a Centricon C-3 microconcentrator (molecular weight cutoff approximately 3000) but was excluded from Sephadex G-10 columns. These parameters enable us to determine an apparent molecular weight of 700-3000 for this factor. The presence of large amounts of this factor in nuclei accounts for the previously unexplained observation that, following size exclusion chromatography, more activated complexes bind to nuclei than to DNA. These data indicate that some, but not all, of the activated complexes require factor to be able to bind to DNA. The predominantly nuclear localization of this factor, coupled with its ability to increase DNA binding, attests to the biological relevance of this factor in the whole cell action of receptor-glucocorticoid complexes.     

Molecular and cytogenetic evidence for cryptic speciation within a rare endemic Malagasy lemur, the Northern Sportive Lemur (Lepilemur septentrionalis)

Evolutionary relationships of different populations of the threatened malagasy lemur Lepilemur septentrionalis were assessed by sequence analysis of mitochondrial DNA (D-loop region and partial Cyt b gene). One hundred and fifty nine samples were collected from five main different localities in the northern part of Madagascar. We applied the phylogenetic species concept based on fixed diagnostic differences to determine the status of different geographical populations. No nucleotide site diagnoses Ankarana from Andrafiamena or Analamera. However, numerous fixed differences separate Sahafary from all other populations. These results were corroborated by phylogenetic trees. As previous cytogenetic studies, our molecular data suggest that two cryptic species of Lepilemur occur in the extreme north of Madagascar. This speciation is probably caused by chromosomal rearrangements in at least one of the evolutionary lineages. Our study comprises another striking example of how molecular genetic assay can detect phylogenetic discontinuities that are not reflected in traditional morphologically based taxonomies. Our study indicates that the Sahafary population is a hitherto undescribed endangered endemic species which urgently needs conservation efforts.     

Amplification of the Y chromosome in three murine tumor cell lines transformed in vivo by different human prostate cancers

Summary Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5–60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells; (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.     

Y chromosome--specific DNA sequences in Turner-syndrome mosaicism.

Phenotypic females with Y-chromosomal material in their genome have an increased risk for development of gonadal malignancy. The detection and identification of Y-chromosomal material in these cases can be of critical importance for medical management. Chromosome analysis in four patients with Turner syndrome revealed the characteristic 45,X chromosome complement together with a second cell population containing a small marker chromosome (46,X, + mar). Molecular-hybridization analyses utilizing cloned, Y chromosome-specific DNA sequences were performed to determine whether Y-chromosomal material was present in each patient. Three cases contained some Y chromosome-specific sequences, whereas one case was negative with all four probes that we used. These results were compared with detailed cytogenetic studies--including G-, Q-, and G-11-banding--of the marker chromosomes. In one case in which Y chromosome-specific DNA sequences were demonstrated, the marker chromosome was G-11 negative. These results demonstrate that cytogenetic analysis alone can lead to misidentification of some Y chromosome-derived markers. The combination of cytogenetic and molecular analyses permits a more accurate characterization of anomalous Y chromosomes and in turn provides additional information that can be crucial to the correct medical management of Turner-syndrome patients.     

Gene mapping in the rat by mouse-rat somatic cell hybridization: synteny of the albumin and alpha-fetoprotein genes and assignment to chromosome 14

The methods of somatic cell genetics and molecular hybridization were applied to a panel of mouse X rat hepatocyte hybrids segregating rat chromosomes to assign the rat genes coding for two serum proteins, albumin and alpha-fetoprotein (Alb and Afp). The molecular hybridization of DNAs from different hybrids with cloned DNA probes showed that all the hybrid clones possessing the rat Alb gene and expressing it also retained the rat Afp locus, which is not expressed in these hybrids. So the Alb and Afp genes are syntenic in the rat, as in the mouse. Furthermore, the cytogenetic analysis allowed the assignment of these two loci to rat chromosome 14.