Banding patterns in Drosophila melanogaster polytene chromosomes correlate with DNA-binding protein occupancy

The most enigmatic feature of polytene chromosomes is their banding pattern, the genetic organization of which has been a very attractive puzzle for many years. Recent genome-wide protein mapping efforts have produced a wealth of data for the chromosome proteins of Drosophila cells. Based on their specific protein composition, the chromosomes comprise two types of bands, as well as interbands. These differ in terms of time of replication and specific types of proteins. The interbands are characterized by their association with "active" chromatin proteins, nucleosome remodeling, and origin recognition complexes, and so they have three functions: acting as binding sites for RNA pol II, initiation of replication and nucleosome remodeling of short fragments of DNA. The borders and organization of the same band and interband regions are largely identical, irrespective of the cell type studied. This demonstrates that the banding pattern is a universal principle of the organization of interphase polytene and non-polytene chromosomes.     

Somatic mosaicism in cases with small supernumerary marker chromosomes

Somatic mosaicism is something that is observed in everyday lives of cytogeneticists. Chromosome instability is one of the leading causes of large-scale genome variation analyzable since the correct human chromosome number was established in 1956. Somatic mosaicism is also a well-known fact to be present in cases with small supernumerary marker chromosomes (sSMC), i.e. karyotypes of 47,+mar/46. In this study, the data available in the literature were collected concerning the frequency mosaicism in different subgroups of patients with sSMC. Of 3124 cases with sSMC 1626 (52%) present with somatic mosaicism. Some groups like patients with Emanuel-, cat-eye- or i(18p)- syndrome only tend rarely to develop mosaicism, while in Pallister-Killian syndrome every patient is mosaic. In general, acrocentric and non-acrocentric derived sSMCs are differently susceptible to mosaicism; non-acrocentric derived ones are hereby the less stable ones. Even though, in the overwhelming majority of the cases, somatic mosaicism does not have any detectable clinical effects, there are rare cases with altered clinical outcomes due to mosaicism. This is extremely important for prenatal genetic counseling. Overall, as mosaicism is something to be considered in at least every second sSMC case, array-CGH studies cannot be offered as a screening test to reliably detect this kind of chromosomal aberration, as low level mosaic cases and cryptic mosaics are missed by that.     

Regulation of bacteriophage Mu transposition

Bacteriophage Mu is a transposon and a temperate phage which has become a paradigm for the study of the molecular mechanism of transposition. As a prophage, Mu has also been used to study some aspects of the influence of the host cell growth phase on the regulation of transposition. Through the years several host proteins have been identified which play a key role in the replication of the Mu genome by successive rounds of replicative transposition as well as in the maintenance of the repressed prophage state. In this review we have attempted to summarize all these findings with the purpose of emphasizing the benefit the virus and the host cell can gain from those phage-host interactions.     

Microdissection of plant chromosomes by argon-ion laser beam

Summary Rice and barley chromosomal samples were prepared both on a polyester membrane and on an ordinary glass slide and subjected to microdissection by an argon-ion laser. The intensity and the position of the laser beam were controlled by a microcomputer. The most suitable intensity to obtain chromosomal fragments was determined experimentally. As a result, specific regions of the centromere, satellite, short arm, or long arm, of the barley and rice chromosomes were dissected out from the chromosomal spreads. Chromosomal fragments were also successfully transferred from the sample into an Eppendorf tube.     

Strategies for engineering human chromosomes with therapeutic potential

Human engineered chromosomes (HECs) have several potential advantages over currently used vectors for gene therapy applications. Firstly, there is no upper size limit to DNA that can be cloned in these vectors. Secondly, their extrachromosomal nature ensures that introduced genes are neither disruptive to, nor affected by, the genome of the host cell. Finally, being solely human in origin, HEC vectors should not evoke adverse host immunogenic responses. Recent advances have produced a variety of HECs via several different approaches. This review focuses on the current methodologies for making HEC vectors, the advantages and problems associated with each strategy, and discusses the outlook for HEC vectors as ex vivo therapeutic agents.     

Human sperm chromosomes in zona-free hamster eggs: Pretreatment of spermatozoa with calcium ionophore facilitates later examination of chromosomes

Freshly ejaculated human spermatozoa exposed to 10 μM calcium ionophore A23187 for 30 minutes quickly bound to and penetrated into zona-free hamster eggs. When these eggs were cultured for 13 hours in the presence of low concentrations of vinblastin and podophyllotoxin, then fixed and spread with the use of a cold fixation technique, haploid sets of sperm chromosomes were demonstrated. The number of spermatozoa that were attached to the egg surface at insemination influenced the success rate of chromosome demonstration. The best results (an average of about 0.7 chromosome set per egg) were obtained when insemination was controlled so that an average of seven spermatozoa attached to each egg at the end of a 15–30-minute insemination period. This improved method for demonstrating human sperm chromosomes would be useful in the analysis of paternal genetic contributions in reproduction as well as in assessing the effects of environmental genotoxic agents or conditions on spermatozoa.     

Double minute chromosomes in mouse methotrexate-resistant cells studied by atomic force microscopy

Double minute chromosomes (DMs) are acentric, autonomously replicating extra-chromosomes and frequently mediate gene amplification in tumor and drug resistant cells. Atomic force microscopy (AFM) is a powerful tool in microbiology. We used AFM to explore the ultrastructure of DMs in mouse fibroblasts 3T3R500. DMs in various phases of cell cycle were also studied in order to elucidate the mechanisms of their duplication and separation. Metaphase spread and induced premature condensed chromosomes (PCCs) were observed under the AFM. DMs were detected to be composed of two compact spheres linked by fibers. The fibers of DMs directly connected with metaphase chromosomes were observed. Many single-minutes and few DMs were detected in G1 PCCs, while more DMs were detected in S PCCs than in G1 PCCs. Besides, all of the DMs in G2 PCCs were coupled. Our present results suggested that DMs might divide into single-minutes during or before G1-phase, followed by duplication of the single-minutes in S-phase. Moreover, we introduced a new powerful tool to study DMs and got some ideal results.     

Histone fractionation by high-performance liquid chromatography

A method for the rapid chromatography of histones by high-performance liquid chromatography (HPLC) using a reverse-phase μBondapak C₁₈ column containing a packing of octadecylsilane chemically bonded to silica and a linear elution gradient running from water to acetonitrile is described. Two conditions were found to be necessary to achieve histone fractionation: (i) silylation of the active groups of the silica solid support, and (ii) trifluoroacetic acid (TFA) in the eluting solvents. Greater than 90% of the total [³H]lysine-labeled protein applied to the column was eluted from the column. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The HPLC histone fractions (identified by their electrophoretic mobilities) were eluted from the column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A + H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less hydrophobic and more hydrophobic histone variants). Phosphorylated histone species were not resolved from their unmodified parental species. The volatile nature of the water/acetonitrile/TFA eluting solvent facilitated the recovery of salt-free histones from the eluted HPLC fractions by simple lyophilization. This system is very useful for the rapid isolation of the lysine-rich histones, H1 and H2B, and the variants of histone H3. With further development, this system is expected to extend the advantages of HPLC to the fractionation of histone H4 and the variants of histone H2A as well.     

Positive and negative selection on mammalian Y chromosomes

Y chromosomes are genetically degenerate in most organisms studied. The loss of genes from Y chromosomes is thought to be due to the inefficiency of purifying selection in nonrecombining regions, which leads to the accumulation of deleterious mutations via the processes of hitchhiking, background selection, and Muller's ratchet. As the severity of these processes depends on the number of functional genes linked together on the nonrecombining Y, it is not clear whether these processes are still at work on the old, gene-poor mammalian Y chromosomes. If purifying selection is indeed less efficient in the Y-linked, compared to the X-linked genes, deleterious nonsynonymous substitutions are expected to accumulate faster on the Y chromosome. However, positive selection on Y-linked genes could also increase the rate of amino acid-changing substitutions. Thus, the previous reports of an elevated nonsynonymous substitution rate in Y-linked genes are still open to interpretation. Here, we report evidence for positive selection in two out of three studied mammalian Y-linked genes, suggesting that adaptive Darwinian evolution may be common on mammalian Y chromosomes. Taking positive selection into account, we demonstrate that purifying selection is less efficient in mammalian Y-linked genes compared to their X-linked homologues, suggesting that these genes continue to degenerate.     

Structure of mitotic chromosomes

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Why plant chromosomes do not show G-bands

Summary Giemsa techniques have refused to reveal G-banding patterns in plant chromosomes. Whatever has been differentially stained so far in plant chromosomes by various techniques represents constitutive heterochromatin (redefined in this paper). Patterns of this type must not be confused with the G-banding patterns of higher vertebrates which reveal an additional chromosome segmentation beyond that due to constitutive heterochromatin. The absence of G-bands in plants is explained as follows: 1) Plant chromosomes in metaphase contain much more DNA than G-banding vertebrate chromosomes of comparable length. At such a high degree of contraction vertebrate chromosomes too would not show G-bands, simply for optical reasons. 2) The striking correspondence of pachytene chromomeres and mitotic G-bands in higher vertebrates suggests that pachytene chromomeres are G-band equivalents, and that this may also be the case in plants. G-banded vertebrate chromosomes are on the average only 2.3 times shorter in mitosis than in pachytene; the chromomeric pattern therefore still can be shown. In contrast, plant chromosomes are approximately 10 times shorter at mitotic metaphase; their pachytene-like arrangement of chromomeres is therefore no longer demonstrable.     

Modulation of DNA conformations through the formation of alternative high-order HU-DNA complexes

HU is an abundant, highly conserved protein associated with the bacterial chromosome. It belongs to a small class of proteins that includes the eukaryotic proteins TBP, SRY, HMG-I and LEF-I, which bind to DNA non-specifically at the minor groove. HU plays important roles as an accessory architectural factor in a variety of bacterial cellular processes such as DNA compaction, replication, transposition, recombination and gene regulation. In an attempt to unravel the role this protein plays in shaping nucleoid structure, we have carried out fluorescence resonance energy transfer measurements of HU-DNA oligonucleotide complexes, both at the ensemble and single-pair levels. Our results provide direct experimental evidence for concerted DNA bending by HU, and the abrogation of this effect at HU to DNA ratios above about one HU dimer per 10-12 bp. These findings support a model in which a number of HU molecules form an ordered helical scaffold with DNA lying in the periphery. The abrogation of these nucleosome-like structures for high HU to DNA ratios suggests a unique role for HU in the dynamic modulation of bacterial nucleoid structure.     

Photographic map of the polytene chromosomes of Cochliomyia hominivorax

Abstract.  Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. A polytene chromosome map is an invaluable tool for the genetic analysis and manipulation of any species because it allows the integration of physical and genetic maps. Cochliomyia hominivorax has a diploid number of 12 chromosomes (2 n  = 12): five pairs of autosomes and one pair of sex chromosomes (XX/XY), which do not polytenize. We created a new photomap of the polytene chromosomes of C. hominivorax describing its five autosomes (chromosomes 2–6). Pupal trichogen cells, which have chromosomes with a high degree of polytenization, were used to elaborate this map. The photomap was made by comparing 20 different nuclei and choosing, for each chromosome segment, the region with the highest resolution. Thus, we present a new photomap of the five autosomes of this species, with a total resolution of 1450 bands.     

Conservation of sex chromosomes in lacertid lizards

Sex chromosomes are believed to be stable in endotherms, but young and evolutionary unstable in most ectothermic vertebrates. Within lacertids, the widely radiated lizard group , sex chromosomes have been reported to vary in morphology and heterochromatinization, which may suggest turnovers during the evolution of the group. We compared the partial gene content of the Z‐specific part of sex chromosomes across major lineages of lacertids and discovered a strong evolutionary stability of sex chromosomes. We can conclude that the common ancestor of lacertids, living around 70 million years ago (Mya), already had the same highly differentiated sex chromosomes. Molecular data demonstrating an evolutionary conservation of sex chromosomes have also been documented for iguanas and caenophidian snakes. It seems that differences in the evolutionary conservation of sex chromosomes in vertebrates do not reflect the distinction between endotherms and ectotherms, but rather between amniotes and anamniotes, or generally, the differences in the life history of particular lineages.     

Satellite DNA and chromosomes in Neotropical fishes: methods,applications and perspectives

Constitutive heterochromatin represents a substantial portion of the eukaryote genome, and it is mainly composed of tandemly repeated DNA sequences, such as satellite DNAs, which are also enriched by other dispersed repeated elements, including transposons. Studies on the organization, structure, composition and in situ localization of satellite DNAs have led to consistent advances in the understanding of the genome evolution of species, with a particular focus on heterochromatic domains, the diversification of heteromorphic sex chromosomes and the origin and maintenance of B chromosomes. Satellite DNAs can be chromosome specific or species specific, or they can characterize different species from a genus, family or even representatives of a given order. In some cases, the presence of these repeated elements in members of a single clade has enabled inferences of a phylogenetic nature. Genomic DNA restriction, using specific enzymes, is the most frequently used method for isolating satellite DNAs. Recent methods such as C₀t–1 DNA and chromosome microdissection, however, have proven to be efficient alternatives for the study of this class of DNA. Neotropical ichthyofauna is extremely rich and diverse enabling multiple approaches with regard to the differentiation and evolution of the genome. Genome components of some species and genera have been isolated, mapped and correlated with possible functions and structures of the chromosomes. The 5SHindIII‐DNA satellite DNA, which is specific to Hoplias malabaricus of the Erythrinidae family, has an exclusively centromeric location. The As51 satellite DNA, which is closely correlated with the genome diversification of some species from the genus Astyanax, has also been used to infer relationships between species. In the Prochilodontidae family, two repetitive DNA sequences were mapped on the chromosomes, and the SATH 1 satellite DNA is associated with the origin of heterochromatic B chromosomes in Prochilodus lineatus. Among species of the genus Characidium and the Parodontidae family, amplifications of satellite DNAs have demonstrated that these sequences are related to the differentiation of heteromorphic sex chromosomes. The possible elimination of satellite DNA units could explain the genome compaction that occurs among some species of Neotropical Tetraodontiformes. These topics are discussed in the present review, showing the importance of satellite DNA analysis in the differentiation and karyotype evolution of Actinopterygii.     

Translocation of histone proteins across lipid bilayers and Mycoplasma membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).     

High-resolution HKG-banding in maize mitotic chromosomes

Root-tip maize chromosomes (2n=20) were prepared by high-resolution procedures after ethidium bromide/colchicine synchronization. Using HKG-banding (HCl−KOH-Giemsa), that shows both centromeric and intercalary heterochromatin, the banding pattern of the elongated-chromosomes showed one to nine well-resolved dark bands. Differences of HKG-banding pattern of elongated and compacted chromosomes were performed by image analysis.     

Some observations on the structure, cation content and possible evolutionary status of dinoflagellate chromosomes

SICEE, D. C., 1984. Some observations on the structure, cation content and possible evolutionary status of dinoflagellate chromosomes. Dinoflagellate chromosomes have a well-ordered structure, as observed in living cells, glutaraldehyde/osmium tetroxide-fixed cells, ultrathin cryosections and freeze-etch preparations. It is suggested that the stabilization of this chromatin in the living cell is largely mediated by divalent cations, acting as bridging molecules between the DNA superstructure and the protein matrix. Studies using X-ray micro-analysis and autoradiography have shown that these chromosomes have high levels of bound Ca and transition metals, and that these are associated with both the DNA and surrounding proteins.
The organization and stabilization of chromatin in dinoflagellate chromosomes is quite different from that of the cells of other eukaryotes, but shows some resemblance to the dispersed chromatin of bacteria. The evolution of dinoflagellate chromosomes from a prokaryote-like ancestral genome is attributed to two main factors–the retention of a primitive cationic non-histone stabilization system, and a pronounced evolutionary trend towards high DNA values. On this theory, dinoflagellate chromosomes are phylogenetically distinct from all other eukaryote chromosomes, and provide a separate evolutionary route for the attainment of high DNA levels and increased cell size.