Chromosome Class Research Articles

An Open Dataset of Annotated Metaphase Cell Images for Chromosome Identification

Chromosomes are a principal target of clinical cytogenetic studies. While chromosomal analysis is an integral part of prenatal care, the conventional manual identification of chromosomes in images is time-consuming and costly. This study developed a chromosome detector that uses deep learning and that achieved an accuracy of 98.88% in chromosomal identification. Specifically, we compiled and made available a large and publicly accessible database containing chromosome images and annotations for training chromosome detectors. The database contains five thousand 24 chromosome class annotations and 2,000 single chromosome annotations. This database also contains examples of chromosome variations. Our database provides a reference for researchers in this field and may help expedite the development of clinical applications.

Classification of fluorescent R-Band metaphase chromosomes using a convolutional neural network is precise and fast in generating karyograms of hematologic neoplastic cells

Karyotype analysis has a great impact on the diagnosis, treatment and prognosis in hematologic neoplasms. The identification and characterization of chromosomes is a challenging process and needs experienced personal. Artificial intelligence provides novel support tools. However, their safe and reliable application in diagnostics needs to be evaluated. Here, we present a novel laboratory approach to identify chromosomes in cancer cells using a convolutional neural network (CNN). The CNN identified the correct chromosome class for 98.8% of chromosomes, which led to a time saving of 42% for the karyotyping workflow. These results demonstrate that the CNN has potential application value in chromosome classification of hematologic neoplasms. This study contributes to the development of an automatic karyotyping platform.

Protease‐deficient SOS constitutive cells have RecN‐dependent cell division phenotypes

In Escherichia coli, after DNA damage, the SOS response increases the transcription (and protein levels) of approximately 50 genes. As DNA repair ensues, the level of transcription returns to homeostatic levels. ClpXP and other proteases return the high levels of several SOS proteins to homeostasis. When all SOS genes are constitutively expressed and many SOS proteins are stabilized by the removal of ClpXP, microscopic analysis shows that cells filament, produce mini-cells and have branching protrusions along their length. The only SOS gene required (of 19 tested) for the cell length phenotype is recN. RecN is a member of the Structural Maintenance of Chromosome (SMC) class of proteins. It can hold pieces of DNA together and is important for double-strand break repair (DSBR). RecN is degraded by ClpXP. Overexpression of recN+ in the absence of ClpXP or recN4174 (A552S, A553V), a mutant not recognized by ClpXP, produce filamentous cells with nucleoid partitioning defects. It is hypothesized that when produced at high levels during the SOS response, RecN interferes with nucleoid partitioning and Z-Ring function by holding together sections of the nucleoid, or sister nucleoids, providing another way to inhibit cell division.

Updating the maize karyotype by chromosome DNA sizing.

The karyotype is a basic concept regarding the genome, fundamentally described by the number and morphological features of all chromosomes. Chromosome class, centromeric index, intra- and interchromosomal asymmetry index, and constriction localization are important in clinical, systematic and evolutionary approaches. In spite of the advances in karyotype characterization made over the last years, new data about the chromosomes can be generated from quantitative methods, such as image cytometry. Therefore, using Zea mays L., this study aimed to update the species’ karyotype by supplementing information on chromosome DNA sizing. After adjustment of the procedures, chromosome morphometry and class as well as knob localization enabled describing the Z. mays karyotype. In addition, applying image cytometry, DNA sizing was unprecedentedly measured for the arms and satellite of all chromosomes. This way, unambiguous identification of the chromosome pairs, and hence the assembly of 51 karyograms, were only possible after the DNA sizing of each chromosome, their arms and satellite portions. These accurate, quantitative and reproducible data also enabled determining the distribution and variation of DNA content in each chromosome. From this, a correlation between DNA amount and total chromosome length evidenced that the mean DNA content of chromosome 9 was higher than that of chromosome 8. The chromosomal DNA sizing updated the Z. mays karyotype, providing insights into its dynamic genome with regards to the organization of the ten chromosomes and their respective portions. Considering the results and the relevance of cytogenetics in the current scenario of comparative sequencing and genomics, chromosomal DNA sizing should be incorporated as an additional parameter for karyotype definition. Based on this study, it can be affirmed that cytogenetic approaches go beyond the simple morphological description of chromosomes.

Fully Unsupervised M-FISH Chromosome Image Characterization

Chromosome analysis is an important and difficult task for clinical diagnosis and biological research. A color imaging technique, multiplex fluorescent in situ hybridization (M-FISH), has been developed to ease the analysis of the process. Using an M-FISH technique each chromosome class (1,2, …,22,X,Y) is stained with a unique color. However, significant variations between images are observed due to a number of factors such as uneven hybridization and spectral overlap among channels. These types of variations influence the pixel classification accuracy of image classification methods which are supervised and require a set of annotated images for training. In this paper, we present a fully unsupervised M-FISH chromosome image classification methodology. Our main contributions are 1) the assumption that the intensity of a chromosome pixel is sampled from multiple Gaussian components [Gaussian mixture model (GMM)] such that each component corresponds to one chromosome class, and 2) the initialization of the GMM model using the emission information of each chromosome class. This is feasible since prior to the M-FISH image acquirement, we already know which chromosome class is emitting to each of the five M-FISH image channels. The method has been tested on a large number of M-FISH images and an overall accuracy of 89.85% is reported. Our method is unsupervised and presents higher classification accuracy even when it is compared with common supervised based methods. Since the developed classification method does not require training data, it is highly convenient when ground truth does not exist.

Prevalence and Clinical Implications of N-RAS Mutations in Childhood AML – A Report From the Children's Oncology Group.

Abstract 3115Poster Board III-52Mutations in the RAS proto-oncogenes are frequent in acute myeloid leukemia (AML) and serve as prototypic Class I lesions, initiating key downstream hyper-proliferative signal transduction pathways. N-RAS mutations (N-RASmut) are common in AML, occurring in 10-20% of adult and pediatric AML patients; however their prognostic significance in both adults and children remains disputed. Due to a frequent association with a normal karyotype, delineating the impact of these mutations on outcome may enable appropriate risk-adapted therapeutic approaches. Here, we report on the incidence and prognostic significance of N-RASmut in a cohort of 825 pediatric AML patients treated on 2 recent co-operative group studies, CCG-2961 and COG AAML03P1. In total, of the 825 children with de novo AML who underwent N-RAS mutational analysis, 86 (10%) were positive. Gender, age, race, presence of hepatosplenomegaly and FAB subtype were comparable between patients with and without N-RASmut. There was no statistically significant difference between those with and without N-RASmut with respect to specific chromosome class. FLT3/Internal tandem duplications (FLT3/ITD) were less common in those with N-RASmut (2% vs. 9%, p=0.03). In contrast, nucleophosmin (NPM) mutations were more common in those with N-RASmut (13% vs. 5%, p=0.02). Overall, N-RASmut showed a significant correlation with low risk AML (CBF, CEBPαa, or NPM, p=0.04) and an inverse relationship with high risk disease (-5/5q- or -7, FLT3/ITD/high allelic ratio, p=0.007). Patients with N-RASmut had similar complete remission (CR) rates following two courses of induction chemotherapy compared with non-mutated patients (79% vs. 79%, p=0.92). Those in CR had a similar relapse rate regardless of the presence of N-RASmut (RR 39% vs. 36%, p=0.97). Five year event free survival (EFS) and overall survival (OS) from study entry were also comparable, however N-RASmut patients demonstrated a marked increase in overall treatment related mortality (TRM) (21% vs. 14%, p=0.03), which was maintained when high and low risk patients were excluded (22% vs. 11%, p=0.05). Evaluation of TRM in CCG-2961 compared with COG AAML03P1 demonstrated that increased TRM in N-RASmut was only seen in patients treated on CCG-2961 and TRM was primarily due to invasive fungal and gram negative infections. Interestingly, for patients treated on CCG-2961, who received interval compressed intensive induction therapy, this increase in TRM correlated with a decrease in both OS and disease free survival from CR. Remission specimens from patients with N-RASmut were negative for the mutation, demonstrating that an increased infectious risk could not be attributed to a host polymorphism. We found N-RASmut did not contribute to increased disease recurrence in pediatric AML. Our findings indicate that N-RAS mutations do not identify a high risk population, however, as such mutations lead to downstream activation of signal transduction pathways, they may identify a target for directed therapy. DisclosuresNo relevant conflicts of interest to declare.

A Multichannel Watershed-Based Segmentation Method for Multispectral Chromosome Classification

Multiplex fluorescent in situ hybridization (M-FISH) is a recently developed chromosome imaging technique where each chromosome class appears to have a distinct color. This technique not only facilitates the detection of subtle chromosomal aberrations but also makes the analysis of chromosome images easier; both for human inspection and computerized analysis. In this paper, a novel method for segmentation and classification of M-FISH chromosome images is presented. The segmentation is based on the multichannel watershed transform in order to define regions of similar spatial and spectral characteristics. Then, a Bayes classifier, task-specific on region classification, is applied. Our method consists of four basic steps: 1) computation of the gradient magnitude of the image, 2) application of the watershed transform to decompose the image into a set of homogenous regions, 3) classification of each region, and 4) merging of similar adjacent regions. The method is evaluated using a publicly available chromosome image database and the obtained overall accuracy is 82.4%. By introducing the classification of each watershed region, the proposed method achieves substantially better results compared to other methods at a lower computational cost. The combination of the multichannel segmentation and the region-based classification is found to improve the overall classification accuracy compared to pixel-by-pixel approaches.

Regulation of Leydig cell steroidogenesis by extracellular signal-regulated kinase 1/2: role of protein kinase A and protein kinase C signaling

The steroidogenic acute regulatory (StAR) protein plays a central role in the regulation of steroid biosynthesis. While steroidogenesis is influenced by many processes, their modes of actions, in a few cases, remain obscure. In this study, we explored the mechanism of action of one such signaling pathway, the extracellular signal-regulated kinase 1/2 (ERK1/2), in regulating StAR expression and steroidogenesis in conjunction with the protein kinase A (PKA) and protein kinase C (PKC) pathways. Using MA-10 mouse Leydig tumor cells, we demonstrate that the activation of PKC and PKA signaling, by phorbol-12-myristate-13-acetate (PMA) and dibutyryl cAMP (dbcAMP)/human chorionic gonadotropin (hCG) respectively, was able to phosphorylate ERK1/2, an event markedly decreased by an upstream kinase inhibitor, U0126. Treatment with PMA enhanced StAR protein expression (associated with a slight increase in progesterone synthesis) but not its phosphorylation (P-StAR), which, in contrast, coordinately increased in response to dbcAMP/hCG. Inhibition of ERK1/2 activity by U0126 decreased PMA-treated StAR expression but increased dbcAMP/hCG-mediated StAR and P-StAR; however, progesterone levels were attenuated. U0126 was found to affect StAR expression and steroidogenesis both at the transcriptional and translational levels. Further studies demonstrated that the effect of U0126 on PMA- and dbcAMP/hCG-mediated StAR expression and steroid synthesis was tightly correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) and scavenger receptor class B type 1 (SR-B1). In fact, both DAX-1 and SR-B1 appear to play important roles in hormone-regulated steroidogenesis. These findings clearly demonstrate that the ERK1/2 signaling cascade involved in regulating StAR expression and steroid synthesis is mediated by multiple factors and pathways and is stimulus specific in mouse Leydig cells.

CAMP-independent signaling regulates steroidogenesis in mouse Leydig cells in the absence of StAR phosphorylation

In the regulation of steroid biosynthesis, a process mediated by the steroidogenic acute regulatory (StAR) protein, both cAMP-dependent and -independent pathways are involved. While the cAMP-dependent regulatory events represent, by far, the most robust increase in steroid synthesis and are well established, the knowledge regarding cAMP-independent mechanisms is lacking. The present investigation was designed to elucidate the potential involvement of the latter in regulating StAR expression and steroidogenesis in mouse Leydig tumor cells (mLTC-1 cells). Treatment of mLTC-1 cells with a number of factors including insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), fibroblast growth factor, transforming growth factor (TGF)alpha, interleukin-1 (IL-1), and colony-stimulating factor-1, increased the levels of StAR mRNA, StAR protein, and progesterone to varying degrees and utilized signaling pathways that are not associated with elevations in intracellular cAMP levels. Importantly, phosphorylation of StAR in response to these stimuli was undetectable, which is in marked contrast to observations with human chorionic gonadotropin (hCG), indicating factors that do not alter intracellular cAMP, regulate the steroid biosynthesis in a StAR phosphorylation-independent manner. In addition, the roles for factors involved in cross-talk between the protein kinase pathways, PKA and PKC, were demonstrated. Further characterization of signaling by one such cAMP-independent factor, TGFalpha, demonstrated that the mechanism, whereby it increased StAR expression and steroid synthesis, was dependent on de novo protein synthesis and mediated via activation of the EGF receptor. TGFalpha was also able to augment hCG-stimulated cAMP synthesis, StAR protein and StAR phosphorylation, and influence hCG binding and LH receptor mRNA expression. Furthermore, TGFalpha increased phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and cAMP-response element-binding protein (CREB), processes inhibited by the mitogen-activated protein kinase/ERK inhibitor U0126 and by expression of non-phosphorylatable CREB-M1 respectively. Inhibition of ERK activity enhanced TGFalpha-mediated StAR protein expression (but not its phosphorylation) and decreased progesterone synthesis, events correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) and scavenger receptor class B type 1 (SR-B1). Collectively, these findings demonstrate that, in mouse Leydig cells, cAMP-independent signaling events regulate steroidogenesis in a StAR phosphorylation-independent manner.

Molecular Mechanisms of Insulin-like Growth Factor-I Mediated Regulation of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells

Growth factors are known to play diverse roles in steroidogenesis, a process regulated by the mitochondrial steroidogenic acute regulatory (StAR) protein. The mechanism of action of one such growth factor, IGF-I, was investigated in mouse Leydig tumor (mLTC-1) cells to determine its potential role in the regulation of StAR expression. mLTC-1 cells treated with IGF-I demonstrated temporal and concentration-dependent increases in StAR expression and steroid synthesis. However, IGF-I had no effect on cytochrome P450 side-chain cleavage or 3beta-hydroxysteroid dehydrogenase protein levels. IGF-I was capable of augmenting N,O'-dibutyrl-cAMP-stimulated steroidogenic responsiveness in these cells. The steroidogenic potential of IGF-I was also confirmed in primary cultures of isolated mouse Leydig cells. IGF-I increased phosphorylation of ERK1/2, an event inhibited by the MAPK/ERK inhibitors, PD98059 and U0126. Interestingly, inhibition of ERK activity enhanced IGF-I-mediated StAR protein expression, but phosphorylation of StAR was undetectable, an observation in contrast to that seen with N,O'-dibutyrl-cAMP signaling. Further studies demonstrated that these events were tightly correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 and scavenger receptor class B type 1. Whereas both protein kinase A and protein kinase C signaling were involved in the IGF-I-mediated steroidogenic response, the majority of the effects of IGF-I were found to be mediated by the protein kinase C pathway. Transcriptional activation of the StAR gene by IGF-I was influenced by several transcription factors, its up-regulation being dependent on phosphorylation of the cAMP response element-binding protein (CREB) and the activator protein 1 family member, c-Jun. Conversely, StAR gene transcription was markedly inhibited by expression of nonphosphorylatable CREB (Ser(133)Ala), dominant negative A-CREB, and dominant negative c-Jun (TAM-67) mutants. Collectively, the present studies identify molecular events in IGF-I signaling that may influence testicular growth, development, and the Leydig cell steroidogenic machinery through autocrine/paracrine regulation.

Some methods of combining class information in multivariate normal discrimination for the classification of human chromosomes

We consider the use of discriminant analysis based on an assumption of multivariate Normality for allocating human chromosomes in an automated system. In this context, the assumptions which might be made about the covariance matrices for the different chromosome classes have important implications for the error rate of the system and the time required to allocate a chromosome. Linear discriminant functions based on the assumption of a common covariance matrix for all classes are fast but sometimes give bigger error rates than the assumption of a separate covariance matrix for each class. The latter assumption requires many more calculations to evaluate the associated quadratic discriminant functions. However, it is possible to assume that the covariance matrices for the different classes are, in various senses, similar to one another in order to derive other methods of combining class information on variability. These methods are here incorporated in the estimative maximum-likelihood approach to discrimination. The methods considered lead to machine classification times of human chromosomes intermediate between those for the assumptions of a common or unrelated covariance matrices. They also require the simultaneous estimation of fewer parameters than the use of a separate covariance matrix for each chromosome class. The methods are illustrated by three data sets of very different quality. Graphs of estimated error rate against classification time show that some of these ways of combining class information can be useful in the trade-off of error rate against time.

HOMOLOGOUS RECOMBINATION BETWEEN EPISOMAL PLASMIDS AND CHROMOSOMES IN YEAST

We have observed genetic recombination between ura3( -) mutations (among them extensive deletions) carried on "episomal" (i.e., 2micro DNA-containing) plasmids and other ura3( -) alleles present at the normal chromosomal URA3 locus. The recombination frequency found was comparable to the level observed for classical mitotic recombination but was relatively insensitive to sunlamp radiation, which strongly stimulates mitotic recombination. Three equally frequent classes could be distinguished among the recombinants. Two of these are the apparent result of gene conversions (or double crossovers) which leave the URA3(+) allele on the chromosome (class I) or on the plasmid (class II). The third class is apparently due to a single crossover that results in the integration of the plasmid into a chromosome. Plasmid-chromosome recombination can be useful in fine structure genetic mapping, since recombination between a chromosomal point mutation and a plasmid-borne deletion mutation only 25 base pairs distant was easily detected.

The yield and competitive ability of euploid and aneuploid genotypes of tetraploid Lolium perenne in two seasons at two locations

Six genotypes derived from a high and low yielding selection within each of the 3 chromosome classes 27, 28 (euploid) and 29 were grown as pure stands and in all possible binary mixture combinations in a tray experiment carried out at Aberystwyth and Newcastle over a two year period. Dry matter yield, tiller number and plant height were recorded for each of several harvests each season. The results showed that: 1) (i) the euploids were of high yield and average competitive ability, (ii) the hypoploids were of average yield and low competitive ability, (iii) the hyperploids were of lower than average yield and of high competitive ability; 2) Insome cases significant differences were found between selections within a chromosome class; 3) Mixture performance was intermediate to that of the component pure stands; 4) When high yielding selections were grown in a mixture with a selection of superior competitive ability they tended to produce more tillers per unit dry weight than in pure stands. It was concluded that, until more is known of the relative performance of euploids and aneuploids, marketed seed should contain as high a proportion of euploids as possible.

Fast rejoining processes in Nigella damascena chromosomes revealed by fractionated 60Co γ-ray exposures

Dry seeds of Nigella damascena were irradiated by 60Co γ-rays with fractionated exposures (2 × 4 krads), separated by time-intervals ranging from 2 to 3 min with increments of 10 sec. At time-intervals longer than 2 min 10 sec, the frequency of chromosome aberrations was found to be considerably decreased. These fast rejoining processes were oxygen dependent, being suppressed under anoxic conditions. Similarly, respiratory inhibitors: potassium cyanide (1 × 104M), sodium azide (1 × 104M) and dinitrophenol (1 × 105M) suppress the decrease. To test the hypothesis that ionic bonds can be involved in fast rejoining processes, seeds were treated respectively with three chelating agents (at 1 × 104M) before irradiation. Dipyridyl (DP) was found to suppress the decrease whereas diethyldithiocarbamate (DIECA) or cupferron (CUPF) produced a reversion at exactly the same tune-interval. The action of chelating agents was proved to be oxygen dependent. It was also demonstrated that all kinds of aberrations of the chromosome class i.e. breaks, minutes, dicentrics or rings were involved in the processes.

ORIGIN OF FRAGARIA POLYPLOIDS. II. UNREDUCED AND DOUBLED‐UNREDUCED GAMETES

Offspring from natural hybrids between octoploid Fragaria chiloensis (2n = 56) and diploid F. vesca (2n = 14) backcrossed under natural conditions to F. chiloensis were studied. The natural F1 hybrids themselves were of three kinds: (1) The expected pentaploids which resulted from the union of normally reduced gametes of diploid F. vesca and octoploid F. chiloensis; (2) A hexaploid F1 hybrid which resulted from the union of an unreduced gamete from diploid F. vesca with a normally reduced gamete from octoploid F. chiloensis; and (3) A 9‐ploid F1 hybrid which probably arose from the union of an unreduced gamete of the octoploid F. chiloensis with a normally reduced gamete of diploid F. vesca. The progenies that resulted from the natural backcrossing of each of the three sorts of F1 hybrids to F. chiloensis were as follows: The pentaploid F1 hybrids (2n = 35) yielded mostly 9‐ploid offspring from unreduced 5X gametes; a relatively high percentage of 14‐ploid plants arising from doubled‐unreduced 10 X gametes and a few 2N = ±46 aneuploids from reduced gametes. The hexaploid F1 hybrid (2n = 42) on backcrossing yielded over 50% 10‐ploid offspring with the rest 2n = ±50 aneuploids from reduced gametes. The 9‐ploid F1 hybrid (2n = 63) on backcrossing yielded mostly aneuploids normally distributed about a modal 2n = 59 chromosome class resulting from a 31 chromosome gamete, with a few 2n = 56 and 2n = 63 euploids. The 9‐ploids may facilitate diploid Å octoploid introgression. Screening of the open‐pollinated offspring from F. chiloensis revealed almost 2% 12‐ploid (2n = 84) offspring from the union of the reduced and unreduced F. chiloensis gametes. The probable genomic constitution of the observed novel ploidy levels and those that theoretically may be generated from the known hybrids are presented. The origin of the existing polyploids from diploids through simple unreduction is postulated.

Origin of Fragaria Polyploids. II. Unreduced and Doubled-Unreduced Gametes

Offspring from natural hybrids between octoploid Fragaria chiloensis (2n = 56) and diploid F. vesca (2n = 14) backcrossed under natural conditions to F. chiloensis were studied. The natural F1 hybrids themselves were of three kinds: (1) The expected pentaploids which resulted from the union of normally reduced gametes of diploid F. vesca and octoploid F. chiloensis; (2) A hexaploid F1 hybrid which resulted from the union of an unreduced gamete from diploid F. vesca with a normally reduced gamete from octoploid F. chiloensis; and (3) A 9-ploid F1 hybrid which probably arose from the union of an unreduced gamete of the octoploid F. chiloensis with a normally reduced gamete of diploid F. vesca. The progenies that resulted from the natural backcrossing of each of the three sorts of F1 hybrids to F. chiloensis were as follows: The pentaploid F1 hybrids (2n = 35) yielded mostly 9-ploid offspring from unreduced 5X gametes; a relatively high percentage of 14-ploid plants arising from doubled-unreduced 10 X gametes and a few 2N = ±46 aneuploids from reduced gametes. The hexaploid F1 hybrid (2n = 42) on backcrossing yielded over 50% 10-ploid offspring with the rest 2n = ±50 aneuploids from reduced gametes. The 9-ploid F1 hybrid (2n = 63) on backcrossing yielded mostly aneuploids normally distributed about a modal 2n = 59 chromosome class resulting from a 31 chromosome gamete, with a few 2n = 56 and 2n = 63 euploids. The 9-ploids may facilitate diploid Å octoploid introgression. Screening of the open-pollinated offspring from F. chiloensis revealed almost 2% 12-ploid (2n = 84) offspring from the union of the reduced and unreduced F. chiloensis gametes. The probable genomic constitution of the observed novel ploidy levels and those that theoretically may be generated from the known hybrids are presented. The origin of the existing polyploids from diploids through simple unreduction is postulated.

Breakdown and restoration of chromosome stability following inbreeding in a locust.

1. In a sample of 27 males and 15 females from an inbred culture of Pyrgomorpha kraussi four classes of individuals could be distinguished. Those: a) With a standard chromosome complement, 2n = 18 + XX/XY, b) Where the standard complement was supplemented by a single B chromosome, c) Presumably polysomic for structurally altered or unaltered members of the A complement, and d) As in c) but with a B chromosome. 2. Abnormal chromosome behaviour indicative of genotypic unbalance was found in twelve males. 3. Abnormality consists in the occurrence of: a) Polyploid cells, b) Aneuploid cells, c) Chromosome breakage, and d) Unstained regions. The last three anomalies affect only the A chromosomes. 4. There is considerably variation both within and between individuals in respect of these anomalies. But the number of stable males in the B chromosome class is significantly higher than in other classes and all the males with S + B are stable. 5. It is argued that metabolic DNA, and consequently heterochromatic B chromosomes, are important in relation to the mechanical aspects of chromosome behaviour. 6. The B chromosomes are of unknown origin but the extra A chromosomes in aneuploid cells rarely form multivalents and thus one of the conditions of independent evolution is partially fulfilled.