Additional observations on the preparation of R banded human chromosomes with acridine orange.

A number of technical factors which affect acridine orange R banding (RFA banding) were studied. These variables included age of slide, timing of fixation, details of incubation mounting and the use of sequential technics. Optimal RFA banding was obtained between 15 and 20 days but good or very good preparations were obtained between 7 days and 2 months. Improved results were obtained in slides that were 3-4 months old by refixing the slides in ethanol acetic acid. Intermittent movement of slides during incubation in buffer as well as the details of mounting and removal of cover slips were found to be important. The best sequential banding was obtained with the sequence of Q to R but good results were obtained with the sequence G to R using ASG banding. Satisfactory results with the sequence R to C were not obtained. With careful attention to these variables good RFA binding can be obtained over a period of several months.     

Frequencies of centromeric heteromorphisms of human chromosomes 3 and 4 as detected by QFQ technique: can they be identified by RFA technique?

One hundred normal Caucasians were studied by sequential QFQ and RFA banding techniques in order to estimate the type and frequency of heteromorphisms in the centromeric regions of chromosome 3 and 4. Intensity variants were classified into 1 of 5 levels of QFQ banding. QFQ intensity heteromorphisms (greater than or equal to level 3) for chromosomes 3 and 4 were 62 and 15 percent respectively. The interrelationship between QFQ and RFA variants were also examined. When the centromere was brilliant by QFQ, it was found that it was deep red by RFA; when it was pale by QFQ, it was light red by RFA. Neverthless, a blind coded study could not pick up these color variants by RFA. QFQ banding showed variations of the centromeric regions of chromosomes 3 and 4 while RFA banding failed to demonstrate it. It was concluded that QFQ is the most useful technique in detecting the different intensity levels in the centromeric regions of chromsomes 3 and 4.     

Characterization of human heteroploid cell line J-111: Reverse banding patterns of marker chromosomes

The karyotype of the human cell line, J-111, has been studied employing R-banding by fluorescence using acridine orange technique (RFA). The model chromosome number of this line was 112. All human chromosomes except the Y were present in each metaphase. Twenty-one marker chromosomes were distinguished and their possible origins were investigated. Of these, twelve were consistently present in all cells. Nine markers were highly variable. Four typical marker chromosomes of HeLa cells were found and their origins were identified, indicating that the line is a HeLa contaminant. The reverse banding patterns of all marker chromosomes are presented and the value of the RFA technique is discussed.     

Human chromosomal heteromorphisms in american blacks

Summary One hundred normal American Blacks (B) were studied by sequential QFQ and RFA banding techniques in order to estimate the type and frequency of heteromorphisms. Color heteromorphisms were classified into one of six colors by RFA and intensity variation into one of five levels by QFQ. The data are compared with a previously studied Caucasian population (C). The frequencies of QFQ and RFA heteromorphisms were significantly higher in the Black than in the Caucasian population. No racial difference was noted for chromosome 21 by QFQ, while RFA demonstrated a clear difference. It is concluded that the maximum characterization of racial differences of human chromosomal heteromorphisms was far greater by RFA than with QFQ. The present study suggests differences in QFQ and RFA heteromorphisms among the two races.     

Population heteromorphisms of Ag-stained nucleolus organizer regions (NORs) in the acrocentric chromosomes of East Indians

Summary The nucleolar organizer regions (NORs) of acrocentric chromosomes in 70 normal East Indians were examined by Ag-staining (NSG) and acridine orange reverse banding (RFA) techniques. The Ag-stainability of NORs was variable from one individual to another but characteristics were constant within each individual. The average modal number of Agpositive NORs per individual was eight. A racial difference in the expression of NORs is suggested. to study the heteromorphism of NORs, the NSG technique was found to be more useful than RFA. Furthermore, it is concluded that there is no direct relationship between a heteromorphism of NORs identified by NSG and that identified by the RFA technique. Quantitative data on these differences is provided. In addition NOR-regions are classified into five sizes namely; very large, large, medium, small, and very small using subjectively defined criteria.     

Expression of nucleolus organizer regions (NORs) in human acrocentric chromosomes by NSG, QFQ and RFA techniques: are they identical?

The morphology of nucleolus organizer regions (NORs) of human acrocentric chromosomes has been studied in a family by QFQ, RFA, and NSG banding techniques. The pale green colour seen by RFA was found darkly stained by NSG. However, in some instances, NOR regions were expressed by NSG technique but the pale green colour was not seen. Therefore, it is concluded that there is no direct relationship between a heteromorphism identified by one technique and that identified by another.     

A pericentric inversion of a chromosome 4 with a t(4q+10p-) and a familial t(DqDq) in a mentally retarded girl

Summary A complex chromosome abnormality consisting of a pericentric inversion of a chromosome 4, a t(4q+10q-) and a familial t(13q14q) was found in a 12-year-old girl showing minor dysmorphic stigmata, moderate mental retardation and an expressive aphasia. The structural chromosome rearrangements were analyzed by Giemsa (G), quinacrine fluorescence (Q) and terminal acridine orange (T) banding techniques. No loss of chromosome material could be demonstrated to account for the patient's defects.This study was supported by grant No. HD-05221.     

First description of color variations in the annual killifish Millerichthys robustus,and preliminary observations about its geographical distribution

Millerichthys robustus is the only annual killifish distributed in America with phenotypic color variations, not yet described. Accordingly, we first describe the color pattern in both sexes to define its phenotypical variations. We then analyze the frequency of these phenotypes on a geographical scale, in four localities that represent opposite points of Millerichthys’s distribution in the Mexican southeast. Color analysis based on the RGB system allowed us to define five-color phenotypes in males continuously distributed in various perceptual units between two extreme colors (yellow-red): yellow, moderate orange, dark orange, strong orange and red. These color patterns found in M. robustus could be attributed to melanin, carotenoid, and pteridine pigments. The orange phenotypes was present in all localities studied. The yellow phenotype was present only in northeastern and northwestern locations, and the red phenotype was present only in northern populations. Female color variations were observed in the number of ocelli (from 1 to 15) at the base of the caudal peduncle and dorsal region. Ocelli have been associated with anti-predator functions because they resemble the eyes of vertebrates, thus shifting the target of predator attacks to less vital body parts. Females with 3 ocelli were the most frequent phenotype, and females with 13–15 ocelli occurred only in the northern populations. We concluded that male and female of M. robustus are not randomly distributed along their distribution range, which suggest that color phenotypes may react differently to biotic and abiotic factors that probably determine their distribution and frequency within the studied population.

     

Early replication banding reveals a strongly conserved functional pattern in mammalian chromosomes

One of the best documented autosomal linkage associations in man is on chromosome 1p and in the mouse on chromosome 4. On mitotic chromosomes this genetic homology is shown more clearly by early replication banding (RBG; induced by incorporation of 5bromodeoxyuridine (BrdU) in the second half of the S phase) than by structural banding (induced on prefixed chromosomes by denaturation, RFA, or trypsin, GTG). To analyse this phenomenon in more detail, 11 chromosomal regions in man and the domestic cat with known genetic homology were compared. In four chromosome pairs RBG and GTG banding show the same degree of homology. In seven chromosome pairs the homology is more pronounced by RBG than by GTG banding. RFA banding does not reveal the same extent of homology as does RBG banding. These results clearly show a difference between the structural banding pattern, RFA and GTG, and the replication banding pattern, RBG. The following conclusions can be drawn: in chromosomal regions with homologous functions the DNA replicates in the same temporal order. Early replication banding (RBG) reveals a functional pattern in these regions which has been more strongly preserved during evolution than the underlying chromosomal DNA. Differences in chromosomal banding are most prominent in the GTG banding pattern, whereas similarities are most apparent in the RBG banding pattern.     

Q-banding of human chromosomes after BUdR and BCdR treatment

Summary Two different Q patterns were found in BUdR and BCdR treated chromosomes of human lymphocyte cultures: X-type pattern, in which Giemsa and quinacrine banding both are reversed; Y-type pattern, in which Q-banding remains conventional in spite of reverse G-banding. Possible mechanisms of these findings are discussed.     

Early and late replicative chromosomal banding patterns of Gallus domesticus.

Early and late replicating chromosomal banding patterns of Gallus domesticus were investigated by cell synchronization and incorporation of 5'-bromodeoxyuridine during early and late DNA synthesis. The early replicating chromosomal banding patterns observed, as revealed by either acridine orange or Hoechst 33258/propidium iodide staining, were similar to the structural G-banding patterns obtained by trypsin digestion and Giemsa staining. Late replicating chromosomal banding showed extensive reverse band complementarity to the G-banding pattern. Cell synchronization increased the number of prometaphase and metaphase plates available for analysis. G-banding obtained by Hoechst 33258/propidium iodide staining was investigated due to the fact that it is compatible with chromosomal in situ hybridization procedures that use nonisotopically-labeled DNA probes. Standard replicative G-banded and R-banded idiograms, as obtained after cell synchronization, are proposed.     

Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species

Genomes of 11 Quercus species were characterized using cytogenetic (Giemsa C-banding, fluorochrome banding), molecular-cytogenetic (fluorescence in situ hybridization, FISH, to ribosomal genes) and molecular (dot-blot for ribosomal gene-copy number assessment) techniques. Ribosomal genes are the first DNA sequences to be physically mapped in oaks, and the copy number of the 18S-5.8S-26 S rRNA genes is estimated for the first time. Oak karyotypes were analysed on the basis of DAPI banding and FISH patterns; five marker chromosomes were found. In addition, chromosomal organization of ribosomal genes with respect to AT- and GC-differentiated heterochromatin was studied. Fluorochrome staining produced very similar CMA/DAPI banding patterns, and the position and number of ribosomal loci were identical for all the species studied. The 18S-5.8S-26 S rRNA genes in oak complements were represented by a major locus at the subterminal secondary constriction (SC) of the only subtelocentric chromosome pair and a minor locus at paracentromeric SC of one metacentric pair. The only 5 S rDNA locus was revealed at the paracentromeric region of the second largest metacentric pair. A striking karyotypic similarity, shown by both fluorochrome banding and FISH patterns, implies close genome relationships among oak species no matter their geographic origin (European or American) or their ecophysiology (deciduous or evergreens). Dot-blot analysis gave preliminary evidence for different copy numbers of 18S-5.8S-26 S rRNA genes in diploid genomes of Q. cerris, Q. ilex, Q. petraea, Q. pubescens and Q. robur (2700, 1300, 2200, 4000 and 2200 copies, respectively) that was correlated with the size polymorphism of the major locus. Received: 26 February 1999 / Accepted: 16 March 1999     

Traitements discontinus par le BrdU et coloration par l'acridine orange: obtention de marquages R,Q et intermëdiaires

Discontinued treatments with BudR at different periods of the cellular cycle produce various chromosome banding after staining with acridine orange. In particular, it is possible to observe R- or Q- or an intermediary banding, simply by varying the time of incorporation of BudR. This implies that the amount of AT or GC bases present locally in DNA is not directly responsible for the banding observed. Furthermore it appears that a precise correlation exists between replication and R- or Q-banding: the DNA located at each group of bands replicates either early (R-bands) or late (Q-bands). But these timings overlap towards the middle of phase S: if the treatment is given at that time, it is possible to observe aspects intermediary between Q and R.     

Occurrence of chromosomal variations and plant regeneration from long-term-cultured citrus callus

Summary Embryogenic callus of Anliucheng sweet orange (Citrus sinensis Osbeck) is theoretically diploid. However, significant chromosomal variations occurred when the calluses were subcultured and preserved for a long time. Cytological observation revealed a variety of mitotic irregularities underlying the occurrence of chromosomal variations. Despite the ubiquitous existence of chromosomal variations, long-term-cultured calluses were still capable of producing somatic embryos and plants. Interestingly, chromosomal variants were selected against when somatic embryos and plants regenerated from the embryogenic callus. Randomly amplified polymorphic DNA (RAPD) analysis was also carried out to detect DNA sequence variation in regenerated plants derived from the embryogenic callus. No difference in banding patterns was detected. It was clear that the plant regeneration from long-term-cultured callus was inclined to select against somaclonal variations.     

Rapid Identification of Chromosomal Abnormalities in Human Neoplasia

Recent advances in banding techniques have led to the belief that certain chromosomal defects are consistently associated with specific types of human neoplasia. Based on the GTG technique, it has been suggested that the malignant cells of most neoplasias show chromosomal abnormalities (Yunis et al. 1983). From this recent publication of Yunis it appears that the majority of bands involved in carcinogenesis are G-negative, i.e., do not stain by the GTG technique, and it is therefore difficult to localize the breakpoints. In some of our recent publications we emphasized the importance of the RFA technique (Verma and Lubs 1975), which stains Giemsa-negative bands darkly, thus providing precise identification of chromosomal abnormalities (Verma and Dosik 1976). However, this technique cannot be applied until the slides have aged for at least 7 days. Therefore, we are reporting an alternative procedure using BrdU which provides “reverse” banding immediately when the slides are stained with acridness orange and examined with a fluorescence microscope.     

Chromosomes and constitutive heterochromatin distribution in Arctic charr,Salvelinus alpinus (L.) (Pisces: Salmonidae)

The chromosomes, together with their C and Q banding patterns, of Arctic charr from Loch Rannoch, Scotland are described. Q banding revealed fewer bands than C banding and variation between individuals for the number of C bands was found. These results suggest that subsets of heterochromatin exist and that there may be an individual variation in DNA content.     

Studies on somaclonal variation in Phalaenopsis

The morphological and genetic variations in somaclones of Phalaenopsis True Lady “B79-19” derived from tissue culture were evaluated. In 1360 flowering somaclones, no apparent difference was found in the shape of the leaves, whereas flowers in some somaclones were deformed. We have demonstrated that 38 selected random primers can be used to generate amplified segments of genomic DNA and to differentiate polymorphisms of somaclonal variations in Phalaenopsis. The random amplified polymorphic DNA (RAPD) data indicated that normal and variant somaclones are not genetically identical. We also studied the banding patterns of aspartate aminotransferase (AAT) and phosphoglucomutase (PGM) in young leaves of variant and normal somaclones of Phalaenopsis. With respect to AAT, three distinct banding patterns were found in normal somaclones and only two-banded phenotypes were detected in variant somaclones. In a comparison of the banding patterns of PGM isozymes, three to four bands were detected in normal somaclones and two to three bands in variant ones. Received: 15 August 1997 / Revision received: 16 February 1998 / Accepted: 1 May 1998     

Dye plants of East Anatolia Region (Turkey)

Dye plants that are commonly used by the regional people of East Anatolia were studied. The following information was collected: local name of plants, the part of plants containing dye, obtained color, dying substances, and extraction of colors. As a result of area investigations carried out between 1994 and 2000, 50 taxa (used for dying wool yarns in the region) belonging to 38 genera and 26 families were determined from collected specimens By using the dying plants and mediator substances, 15 different colors and tones can be obtained. The main colors are yellow, green, olive green, black, red, blue, dark blue, brown, gray, beige, orange, khaki, mustard, purple, and smoke. The colors and their many different tones were observed on kilims and carpets that are woven in the East Anatolia region of Turkey     

Molecular characterization of cytoplasmic and nuclear genomes in phenotypically abnormal Valencia orange (Citrus sinensis) + Meiwa kumquat (Fortunella crassifolia) intergeneric somatic hybrids

Organelle DNA inheritance of four 10-year-old somatic hybrid trees between Valencia orange [Citrus sinensis (L.) Osbeck] and Meiwa kumquat (Fortunella crassifolia Swingle) was analyzed by cleaved amplified polymorphic sequence (CAPS) and restriction fragment length polymorphisms (RFLPs). Five chloroplast (cp) and three mitochondrial (mt) universal primer pairs were amplified, but no polymorphisms were detected. When the polymerase chain reaction products were digested by 15 restriction enzymes, four polymorphic cpDNA-CAPS and two mtDNA-CAPS markers were found. Both the cpDNA and mtDNA in the somatic hybrids were derived from Valencia orange (the embryogenic suspension parent). Genomic DNA of the somatic hybrids and corresponding parents was digested by five restriction endonucleases and hybridized with one chloroplast probe (RbcL- RbcL) and nine mitochondrial probes (coxI, coxII, c oxIII, c ob, atpA, tyr, proI, atp6 and atp9). The results indicated that three hybrid plants shared one strong cpDNA band with both parents and that the remaining one plant had two additional novel bands besides the shared band, while their mtDNA was identical to that of Valencia orange plus non-parental bands. When data on the mtDNA banding patterns were combined with observations on phenotypic performance in the field, it was found that the more complex mtDNA banding pattern coincided with increased vigor of the plant. The stability of the organelle genomes was studied by extracting the genomic DNA of one hybrid plant at monthly intervals for 1 year and then analyzing it using RFLPs. Before the dieback of the shoots, two fragments of the mtDNA were lost while the cpDNAs remained stable. Ploidy analysis by flow cytometry showed that all of the hybrids were stable tetraploids. Four simple sequence repeat primer pairs were applied to detect microsatellite alleles of the four hybrid plants, both parents and the 12 DNA samples from one plant. The results showed that all hybrids had biparental bands uniformly, which indicated that they had the same nuclear background. These results suggest that the mtDNA pattern is correlated with the phenotypic abnormality of Valencia and kumquat somatic hybrid plants and that nuclear-cytoplasm incompatibility may be the cause of dieback.