Studies on Meiotic Division in Coccidial and Malarial Parasites

SYNOPSIS. Oocysts of Eimeria tenella and E. maxima , incubated in colcemid after removal from the host gut, were better preserved after fixation with acetic alcohol than oocysts fixed directly. Colcemid may delay hardening of the oocyst but does not interfere with meiosis. During meiosis the nucleolus disappears and 10 rod-like chromosomes are formed by condensation of filamentous, beaded chromosomes, which appear in prophase. Two granules which appear on the nuclear membrane at the end of prophase are interpreted as centrioles. The 10 chromosomes align themselves in 2 rows of 5 parallel with, rather than transverse to, the elongate meiotic spindle. After stretching across the spindle as beaded filaments during migration to the poles, the chromosomes condense as a cluster of 5 rods at each pole. Reduction is thus effected in a single phase without duplication of chromosomes or centromeres.
During meiosis in P. gallinaceum oocysts paired, Y-shaped and paired dot-like chromosomes appear among the pigment granules. No duplication is evident.     

The effect of restriction enzyme digestion of human metaphase chromosomes on C-band variants of chromosomes 1 and 9

Human metaphase chromosomes, fixed on slides, have beent treated with 8 different restriction endonucleases and 29 combinations of 2 restriction enzymes prior to staining with Giemsa. The endonucleases AluI and DdeI and the combinations AluI + DdeI, AluI + HaeIII, AluI + HinfI, and AluI + MboI have then been used to digest metaphase chromosomes of nine individuals with C-band variants of chromosomes 1 or 9, obtained by the CBG technique. The restriction enzyme resistant chromatin of the paracentromeric regions of chromosomes 1 and 9 has been measured and compared with the corresponding CBG-bands. The size of the enzyme resistant chromatin regions depend upon the type of enzyme(s) used. Treatment with AluI + MboI was the only digestion that acted differently on different chromosome pairs. However, within one pair of homologous chromosomes, all digestions revealed the same variations as conventional C-banding.     

DNA segregation: Putting chromosomes in their place

Recent studies provide evidence that bacterial chromosomes are replicated by an enzyme factory, the replisome, located at a fixed position at the center of the cell; the fixed replisome could be a major factor in determining chromosome order in the cell, and may provide the force that drives chromosome segregation.     

Different reactivity of Z-DNA antibodies with human chromosomes modified by actinomycin D and 5-bromodeoxyuridine

Summary Antibodies against Z-DNA react with fixed metaphase chromosomes of man and other mammals. Indirect immunofluorescence staining shows that chromosomal segments corresponding to R- and T-bands preferentially fix Z-DNA antibodies. In this work Z-DNA antibodies were used as a probe for DNA conformation in euchromatin of fixed human chromosomes whose condensation or staining were modified by actinomycin D (AMD) and by 5-bromodeoxyuridine (BrdU). Treatments with AMD and BrdU were performed to induce a G-banding by modification of chromosomal segments corresponding to R- and T-bands. Long BrdU treatments were used to induce asymmetrical and partially undercondensed chromosomes by substitution of thymidine in one or both DNA strand. Our results show a clear difference of Z-DNA antibodies reactivity after AMD or BrdU treatment. The G-banding obtained after AMD treatment is not reversed by Z-DNA antibodies staining since these antibodies bind very weakly to the undercondensed R-bands. On the other hand, the G-banding obtained by BrdU is completely reversed giving typical R-banding, as on untreated chromosomes. For asymmetrical chromosomes an R-, T-banding pattern is always observed but there is a decrease of the fluorescence intensity proportional to the degree of BrdU incorporation. We conclude that AMD treatment greatly disturbs Z-DNA antibodies binding suggesting a change in DNA conformation, whereas BrdU treatments do not suppress but only weaken the specific binding of Z-DNA antibodies on R- and T-bands. The direct involvement of thymidine substitution in DNA sequences recognized by Z-DNA antibodies is discussed.     

Molecular organization of DNA in chromosomes

Metaphase chromosomes of Chinese hamster and lampbrush chromosomes of Triturus viridescens were studied for DNA arrangement by polarized fluorescence microscopy and by linear dichroic ratio measurements. Intact chromosomes showed a small but negative polarization (?8%) of fluorescence, while the dichroic ratio was negative and quite appreciable (0.85). On treatment with trypsin or RNase the polarization value increased to about ?34% with no change in the polarization direction. The dichroic ratio on the other hand remained unchanged on such enzyme treatments. These results, along with the data of form dichroism and the change in fluorescence color following any enzyme action, suggest that basically the preexisting DNA arrangement was revealed by these enzymes. The fully enzyme-treated chromosome–dye complexes showed cross-bands. Quantitatively, the highest degree of DNA orientation across the above two chromosomes was in the range of 15–34%. This orientation fits well with a coiled coil-DNA, which is mostly longitudinal in these chromosomes. It is suggested that this longitudinal coil may have occasional defects in DNA structure. Alternatively, this coil may terminate into regions of random DNA and unsupercoiled DNA showing lateral projections, such as loops. This alternating feature of the DNA arrangement would tandemly repeat in either metaphase and lampbrush chromosomes.     

Restriction endonuclease/nick translation of fixed mouse chromosomes: A study of factors affecting digestion of chromosomal DNA in situ

We used a restriction endonuclease/nick translation procedure to study the ability of certain enzymes, known to cleave mouse satellite DNA in solution, to attack satellite DNA in fixed mouse chromosomes. Although AvaII and Sau96I readily attack the mouse major satellite in fixed chromosomes, BstNI and EcoRII do not normally do so, although if the heterochromatin is uncondensed as a result of culture in the presence of 5-azacytidine, BstNI can attack it. No clear evidence was obtained for digestion in situ of the minor satellite of mouse chromosomes by MspI, the only enzyme reported to cleave this satellite. Our results show that the DNA of mouse heterochromatin is not merely not extracted by certain restriction enzymes, but is actually not cleaved by them. Chromatin conformation is therefore shown to be an important factor in determining patterns of digestion of chromosomes by restriction endonucleases.by D. Schweizer     

The Use of Snail Gut Cytase in Fluorescent Banding Studies on Plant Chromosomes

The fluorescent bands produced in chromosomes by quinacrine derivatives, although highly acid-labile, have been shown to be resistant to digestion by snail gut cytase. This enzyme may therefore be used to soften plant root-tips and so facilitate the production of flat, unbroken mitotic-metaphase plates for fluorescent-banding studies. The roots are fixed in 3:1 absolute alcohol:glacial acetic acid then thoroughly washed in water. The washed roots are then dipped in undiluted cytase and digested for either 2 hrs at room temperature or overnight in a domestic refrigerator. Next they are squashed on a slide and stained in 0.5% aqueous quinacrine hydrochloride for 10-15 minutes at pH 6.2. After washing in three changes of water they are mounted in water. The coverslips are sealed on with clear nail varnish.     

Gyrate atrophy of the choroid and retina: assignment of the ornithine aminotransferase structural gene to human chromosome 10 and mouse chromosome 7.

Gyrate atrophy of the choroid and retina is an autosomal recessive, blinding human disease caused by a deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT). Since human OAT cDNA hybridizes to DNA sequences on both human chromosomes 10 and X, a locus coding for OAT enzyme activity may be present on one or both of these human chromosomes. We have used a series of mouse-human somatic cell hybrids, in combination with starch gel electrophoresis and a histochemical stain for OAT enzyme activity, to assign the structural gene for OAT to human chromosome 10. Our results suggest that the human X chromosome does not contain a locus coding for OAT enzyme activity. In addition, we have used a panel of Chinese hamster-mouse hybrids to assign the murine Oat structural gene to mouse chromosome 7. Our findings, combined with recent molecular studies, indicate that human OAT probes specific for chromosome 10 will be useful for the diagnosis and genetic counseling of individuals at risk for gyrate atrophy.     

Patterns of digestion of human chromosomes by restriction endonucleases demonstrated by in situ nick translation

Summary A restriction enzyme-nick translation procedure has been developed for localizing sites of restriction endonuclease action on chromosomes. This method involves digestion of fixed chromosome preparations with a restriction enzyme, nick translation with DNA polymerase I in the presence of biotinylated-dUTP, detection of the incorporated biotin label with streptavidinalkaline phosphatase, and finally staining for alkaline phosphatase. Results obtained on human chromosomes using a wide variety of restriction enzymes are described, and compared with results of Giemsa and Feulgen staining after restriction enzyme digestion. Results of nick translation are not in general the opposite of those obtained with Giemsa staining, as might have been expected. Although the nick translation procedure is believed to give a more accurate picture of the distribution of restriction enzyme recognition sites on chromosomes than Giemsa staining, it is clear that the results of the nick translation experiments are affected by accessibility to the enzymes of the chromosomal DNA, as well as by the extractability of the DNA.     

Induction of R-bands on human chromosomes by TfiI as the consequence of local differences in target richness.

We present a new restriction enzyme-banding pattern on fixed human chromosomes. R-bands are induced by TfiI, an enzyme that cuts DNA at GA(A/T)TC, that is, at HinfI sites having A or T in the central position. Results suggest that regional differences in the frequency of targets are responsible for the effect caused by this enzyme, whereas conformational differences between G- and R-bands would not affect the enzyme action.     

红翅皱膝蝗减数分裂染色体的螺旋与轴结构

用低渗处理和苯酚品红染色,在经过卡诺液(甲醇3∶冰醋酸1)固定和未经固定的红翅皱膝蝗减数分裂染色体上都看到了螺旋结构。观察和测量结果表明,每条染色单体都是由430nm左右的染色线螺旋形成的。由染色线到染色体的压缩率为4∶1。低渗处理后固定的材料经过银染,则显示了染色体轴结构。同样,未经低渗处理直接固定的材料银染时也出现了轴结构。银染的轴结构位于每个染色单体的中央,并贯穿整个染色单体。在光镜下,这个轴并不是直径均一的棒状结构,而似乎是由许多大小相近的颗粒相连而成。本文对染色体结构的有关模型、骨架和轴结构的真实性以及轴和螺旋的关系等问题进行了讨论。     

Direct incorporation of fluorescein-12-dUTP to insect fixed chromosomes by random primed extension.

The use of an in situ random primed system allows the direct incorporation of fluorescein-12-dUTP into fixed insect chromosomes, resulting in a strong fluorescent labelling. While in an orthopteran species (Eyprepocnemis plorans) a relatively uniform labelling of meiotic and mitotic chromosomes is produced, in Tenebrio molitor (Coleoptera) only the chromosomal arms, but not the pericentromeric heterochromatic areas of mitotic chromosomes, show positive labelling by this method. This indicates that the organization of DNA in heterochromatin is distinct from that in the euchromatin in distantly related species, and in such a way that, in some species, the random hexanucleotides are prevented from annealing with the chromosomal denatured DNA or (and) that primer extension by Klenow enzyme is impeded.     

Spatial order as a source of kinetic cooperativity in organized bound enzyme systems.

When enzyme molecules are distributed within a negatively charged matrix, the kinetics of the conversion of a negatively charged substrate into a product depends on the organization of fixed charges and bound enzyme molecules. Organization is taken to mean the existence of macroscopic heterogeneity in the distribution of fixed charge density, or of bound enzyme density, or of both. The degree of organization is quantitatively expressed by the monovariate moments of charge and enzyme distributions as well as by the bivariate moments of these two distributions. The overall reaction rate of the bound enzyme system may be expressed in terms of the monovariate moments of the charge density and of the bivariate moments of charge and enzyme densities. The monovariate moments of enzyme density do not affect the reaction rate. With respect to the situation where the fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by these two types of moments, generates an increase or decrease of the overall reaction rate as well as a cooperativity of the kinetic response of the system. Thus both the alteration of the rate and the modulation of cooperativity are the consequence of a spatial organization of charges with respect to the enzyme molecules. The rate equations have been derived for different types of organization of fixed charges and enzyme molecules, namely, clustered charges and homogeneously distributed enzyme molecules, clustered enzyme molecules and homogeneously distributed charges, clusters of charges and clusters of enzymes that partly overlap, and clusters of enzymes and clusters of charges that are exactly superimposed. Computer simulations of these equations show how spatial molecular organization may modulate the overall reaction rate.     

Asymmetric staining of Ctenomys chromosomes after treatment with AluI restriction nuclease

After treatment with the endonuclease AluI for 6 or 24 h, chromosomes of two populations of the South American rodent Ctenomys presented an asymmetric banding pattern after Giemsa staining. These asymmetric patterns were chromosome specific (each chromosome of a pair showed different banding pattern) but constant from cell to cell and between homologous chromosomes of the populations analysed. The nature of this peculiar staining is discussed in the light of the interaction between endonucleases and DNA in chromatin of fixed chromosomes.     

Immunohistochemical localization of cyclic guanosine monophosphate (cGMP) on mouse fetal chromosomes

In previous immunohistochemistry studies, cyclic guanosine monophosphate (cGMP) has been found in polytene chromosomes of D. melanogaster, cGMP has not been found in mammalian metaphase chromosomes, but this could be due to loss of cGMP during staining. Thus the effect of different fixation techniques on the immunohistochemically detectable cGMP associated with metaphase chromosomes from mouse fetal tissue was examined. In chromosomes from cells fixed in 2% formalin, or unfixed cells dropped on slides preheated to 60 degrees C, there was diffuse cGMP staining. When cells were fixed in methanol:glacial acetic acid, 3:1, no chromosomal cGMP immunofluorescence was observed, whereas chromosomes from cells fixed in methanol:glacial acetic acid, 6:1, had different patterns of cGMP immunofluorescence depending on the temperature of the slides onto which the fixed cells were dropped. On slides prechilled to 4 degrees C, cGMP immunofluorescence outlined the chromosomes; on room temperature slides, faint chromosomal cGMP staining was observed, and on slides preheated to 68 degrees C or room temperature slides blown dry with hot air, the chromosomes had more intense diffuse cGMP immunofluorescence or distinct symmetrical bands of cGMP immunofluorescence. We have demonstrated the presence of cGMP in mammalian metaphase chromosomes. The different patterns of cGMP immunofluorescence observed may reflect variable preservation of chromosomal proteins that have binding sites for cGMP.     

Molecular characterization and chromosome-specific TRAP-marker development for Langdon durum D-genome disomic substitution lines.

The aneuploid stocks of durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husnot) and common wheat (T. aestivum L.) have been developed mainly in 'Langdon' (LDN) and 'Chinese Spring' (CS) cultivars, respectively. The LDN-CS D-genome chromosome disomic substitution (LDN-DS) lines, where a pair of CS D-genome chromosomes substitute for a corresponding homoeologous A- or B-genome chromosome pair of LDN, have been widely used to determine the chromosomal locations of genes in tetraploid wheat. The LDN-DS lines were originally developed by crossing CS nulli-tetrasomics with LDN, followed by 6 backcrosses with LDN. They have subsequently been improved with 5 additional backcrosses with LDN. The objectives of this study were to characterize a set of the 14 most recent LDN-DS lines and to develop chromosome-specific markers, using the newly developed TRAP (target region amplification polymorphism)-marker technique. A total of 307 polymorphic DNA fragments were amplified from LDN and CS, and 302 of them were assigned to individual chromosomes. Most of the markers (95.5%) were present on a single chromosome as chromosome-specific markers, but 4.5% of the markers mapped to 2 or more chromosomes. The number of markers per chromosome varied, from a low of 10 (chromosomes 1A and 6D) to a high of 24 (chromosome 3A). There was an average of 16.6, 16.6, and 15.9 markers per chromosome assigned to the A-, B-, and D-genome chromosomes, respectively, suggesting that TRAP markers were detected at a nearly equal frequency on the 3 genomes. A comparison of the source of the expressed sequence tags (ESTs), used to derive the fixed primers, with the chromosomal location of markers revealed that 15.5% of the TRAP markers were located on the same chromosomes as the ESTs used to generate the fixed primers. A fixed primer designed from an EST mapped on a chromosome or a homoeologous group amplified at least 1 fragment specific to that chromosome or group, suggesting that the fixed primers might generate markers from target regions. TRAP-marker analysis verified the retention of at least 13 pairs of A- or B-genome chromosomes from LDN and 1 pair of D-genome chromosomes from CS in each of the LDN-DS lines. The chromosome-specific markers developed in this study provide an identity for each of the chromosomes, and they will facilitate molecular and genetic characterization of the individual chromosomes, including genetic mapping and gene identification.