Diagrammatic representation for chromosomal mutagenesis studies. I. Karyotypes most similar to that of man

In an attempt to discover which primate species have the karyotypes most similar to that of man, the chromosome numbers, the Nombre Fondamental and the numbers of chromosomal rearrangements separating the karyotypes of many species were considered. In addition to man, 6 species were selected for their karyotypic similarities and 1 for its dissimilarity. A model representing the karyotypes, by 2 diagrams, is proposed for these 8 species. These diagrams characterize each karyotype, as well as the theoretical random distribution of break-points, after the effect of a clastogenic agent. This allows one to make quantitative analyses of any type of chromosomal rearrangement, with objective criteria, and should improve the study of chromosomal mutagenesis, and particularly interspecies comparisons.     

Chromosomal gene transfer during conjugation by Staphylococcus aureus is mediated by transposon-facilitated mobilization.

A chromosomal copy of the transposon Tn551 and a copy coresident on a gentamicin-resistant conjugative plasmid of Staphylococcus aureus resulted in the mobilization of chromosomal genes during filter mating. Gene mobilization was recA dependent and was not restricted to any specific region of the chromosome. Both essential and nonessential genes were transferred.     

Superhelical DNA in Streptococcus sanguis: role in recombination in vivo.

Summary Competent Streptococcus sanguis treated with non-lethal doses of coumermycin Al immediately before or after uptake of radioactive transforming DNA were reduced in their capacity to yield transformants. This treatment did not alter bacterial ability to bind DNA in DNase I-resistant form, nor did it prevent the single-stranded donor DNA-recipient protein complexes formed upon uptake at the surface of the bacteria from translocating to chromosomal sites. Inhibition of transformation by heterospecific DNA was greater than that by homospecific DNA. The reduction in transformant yield was not accompanied by any loss of donor counts incorporated into the recipient chromosome, but rather by a loss of genetic activity of incorporated donor material indicating a failure of genetic integration and degradation of donor DNA as a consequence of coumermycin treatment. The inhibitory effect of coumermycin on transformation was associated with in vivo loss of chromosomal DNA superhelicity. The chromosomal DNA remained intact, however, indicative of inhibition of a gyrase-like enzyme responsible for the maintenance of negative supercoiling of the S. sanguis chromosome. Upon treatment with the drug, a coumermycin-resistant mutant strain showed neither loss of chromosomal superhelicity nor any inhibitory effect on genetic integration of donor DNA. The evidence supports the idea that chromosomal superhelicity promotes genetic recombination in vivo.     

A comparison of the frequency and type of chromosomal abnormalities in human sperm after different sperm capacitation conditions.

Human sperm karyotypes can be prepared after fusion of human sperm with Golden hamster oocytes. Most laboratories use one of two methods of sperm capacitation: incubation of freshly-ejaculated sperm in Biggers, Whitten, and Whittingham (BWW) medium for 5-7 h at 37 degrees C or sperm storage in (N-tris [hydroxymethyl]methyl-2-aminoethanesulfonic acid; 2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid) (TES)-Tris yolk buffer (TYB) for 1-3 days at 4 degrees C. Since there have been conflicting reports as to whether there is a difference in the frequency of structural chromosomal abnormalities between BWW capacitation and storage in TYB for 2 days, we analyzed a larger number of karyotypes (8974) from 136 donors to determine if there was any difference in the frequency or type of chromosomal abnormalities in sperm treated by fresh BWW capacitation, storage in TYB for 1 day (TYB-1), or storage in TYB for 2 days (TYB-2). There was no difference in the frequency of numerical chromosomal abnormalities or sex ratio in any of the three treatment groups. However, there was a significantly increased frequency of structural chromosomal abnormalities after storage in TYB-1 and TYB-2. There was no difference in the frequency or type of structural chromosomal abnormalities after sperm storage in TYB-1 compared to TYB-2.     

A convenient technique to compare the efficiency of promoters in Escherichia coli.

We describe a technique which allows one to insert any promoter in front of the chromosomal malPQ operon. This can be done easily by using only one plasmid, one strain, and two simple selections. Properties of the final chromosomal fusion are such that the level of amylomaltase, the product of the malQ gene, measures quantitatively the efficiency of the inserted promoter. This method was utilized to compare the efficiency of four well-known promoters: lacZp, trp, tac, lambdaPR and three malT activated promoters: malPp, malkP and malEp.     

Population genetics of modifiers of meiotic drive. II. Linkage modification in the segregation distortion system

The conditions are found under which the frequency of a new gene altering the recombination fraction between two loci controlling meiotic drive will increase. When the model mimics the recombination reducing effects of a chromosomal rearrangement, such as an inversion or duplication, then the result is that the frequency of the chromosomal rearrangement in the population will increase. This may help explain the presence of inversions and duplications (or insertions) in the segregation-distorter system in Drosophila. In some cases it is found that if the new gene causes any alteration in the recombination fraction between the two loci, the frequency of the gene will increase.     

Risk of chromosomal abnormalities, with emphasis on live-born offspring of young mothers.

In a large public urban hospital obstetrics service with > 123,000 deliveries in a 10-year period (1980-89), the frequencies (0.12%) of any type of chromosomal abnormality and of trisomy syndromes were analyzed for maternal age-related risk, by logistic regression. Focusing on very young gravidas, we found that in the study period there were 9,332 births (7.5% of all deliveries) to mothers < or = 16 years old. Estimated risks of chromosomal abnormalities among offspring associated with very young maternal age (9-16 years) were similar to those age-associated risks of mothers 20-29 years old. Risks of chromosomal abnormalities increase with advancing maternal age and are independent of ethnicity.     

Minor chromosomal variants and major chromosomal anomalies in couples with recurrent abortion.

One hundred three women with prior histories of recurrent spontaneous abortion and 81 of their mates were karyotyped with Q-banding during 1976-1980. Recurrent abortion was defined as two or more spontaneous pregnancy losses; no couple with a previous malformed fetus or child was included. These cases were reviewed in order to examine the possible contributions of minor polymorphic chromosomal variants and major chromosomal abnormalities to recurrent spontaneous pregnancy loss. Balanced translocations were detected in four women and two men in the study; mosaic X aneuploidy was noted in one woman. Quantitative (1 qh, 9qh, 16qh, Yqh) and qualitative (3c, 4c, 13p, 13s, 14p, 14s, 15p, 15s, 21p, 21s, 22p, 22s) heterochromatic polymorphisms were blindly assessed and compared with a control group. Cases and controls did not differ in the frequency of any qualitative polymorphisms or in the length of any quantitative polymorphism. Thus, while major parental cytogenetic aberrations are significantly associated with fetal wastage, these data suggest that minor polymorphic chromosomal variants do not play an important role in the etiology of recurrent spontaneous abortion.     

Critical target and dose and dose-rate responses for the induction of chromosomal instability by ionizing radiation.

To investigate the critical target, dose response and dose-rate response for the induction of chromosomal instability by ionizing radiation, bromodeoxyuridine (BrdU)-substituted and unsubstituted GM10115 cells were exposed to a range of doses (0.1-10 Gy) and different dose rates (0.092-17.45 Gy min(-1)). The status of chromosomal stability was determined by fluorescence in situ hybridization approximately 20 generations after irradiation in clonal populations derived from single progenitor cells surviving acute exposure. Overall, nearly 700 individual clones representing over 140,000 metaphases were analyzed. In cells unsubstituted with BrdU, a dose response was found, where the probability of observing delayed chromosomal instability in any given clone was 3% per gray of X rays. For cells substituted with 25-66% BrdU, however, a dose response was observed only at low doses (<1.0 Gy); at higher doses (>1.0 Gy), the incidence of chromosomal instability leveled off. There was an increase in the frequency and complexity of chromosomal instability per unit dose compared to cells unsubstituted with BrdU. The frequency of chromosomal instability appeared to saturate around approximately 30%, an effect which occurred at much lower doses in the presence of BrdU. Changing the gamma-ray dose rate by a factor of 190 (0.092 to 17.45 Gy min(-1)) produced no significant differences in the frequency of chromosomal instability. The enhancement of chromosomal instability promoted by the presence of the BrdU argues that DNA comprises at least one of the critical targets important for the induction of this end point of genomic instability.     

Chromosomal localization of several families of repetitive sequences by in situ hybridization.

Four recombinant DNA clones (H1, H7, H12, and H15) carrying low-repetitive human DNA were previously isolated from a human genomic library based on their specificity for chromosome 21 and were studied for their distribution as determined by in situ hybridization. Clone H7 hybridized to the satellite regions of chromosomes 13, 14, 15, 21, and 22 as well as to the centromere region of chromosome 1. Clone H12 hybridized strongly to chromosomes 11 and 17 and the centromere of the X. Clones H1 and H15 had a very widespread distribution throughout the genome. Clone H15 hybridized significantly more to the short arm of chromosome 18 than to any other chromosomal segment. Clone H1 hybridized strongly to the centromere of chromosome 19 and also showed random distribution on all the other human chromosomes. We conclude that these probes appear to represent four repetitive families that demonstrate in situ hybridization patterns that do not correspond with those of any other repetitive family. Further, the in situ hybridization patterns do not show the strong chromosome 21 specificity originally defined by Southern blot analysis. The nature and chromosomal localization of these repetitive families should be useful in regional mapping and evolutionary studies and give additional insight into chromosomal organization.     

Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria.

Mobile group II introns can be retargeted to insert into virtually any desired DNA target. Here we show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1-22%. Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations. Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria.     

Ribosomal DNA magnification in Saccharomyces cerevisiae.

Strains monosomic for chromosome I of Saccharomyces cerevisiae contain 25 to 35% fewer rRNA genes than do normal diploid strains. When these strains are repeatedly subcultured, colonies are isolated that have magnified their number of rRNA genes to the diploid amount while remaining monosomic for chromosome I. We have determined the amount of DNA complementary to rRNA in viable haploid spores derived from a magnified monosomic strain. Some of these haploids contained 24 to 48% more rRNA genes than a normal euploid strain. These extra genes may be responsible for the increased number of rRNA genes in the strain monosomic for chromosome I. Genetic analysis of the haploids containing extra rRNA genes suggested that these genes are linked to chromosomal DNA and are heterozygous. They were not closely linked to any centromere and were not located on chromosome I. Furthermore, all the DNA complementary to rRNA in one of these haploid strains with magnified rRNA genes sedimented at a chromosomal molecular weight, consistent with chromosomal linkage. In addition, several new mutations mapping on chromosome I were used to show that ribosomal DNA magnification was not due to a chromosome I duplication.     

Prognostic implications of chromosomal findings in acute lymphoblastic leukaemia at diagnosis.

Chromosomes were studied on diagnostic bone-marrow samples from 39 children with acute lymphoblastic leukaemia (ALL). The patients were classified, according to the chromosomal characteristics of the major proportion of their leukaemia cells, into five categories; hyperdiploid, pseudodiploid, diploid, hypodiploid, and mixed. Patients in the hyperdiploid category had significantly longer first remissions than those in all other categories, and those in the pseudodiploid category had the shortest. Neither the absence of any normal cells nor the presence of detectable clones appeared to be an adverse feature. We suggest that the proportion of hyperdiploid cells, determined by conventional chromosomal staining techniques, may be used as an additional prognostic feature in childhood ALL.     

Consecutive chromosomal studies in patients with myelodysplastic syndrome (MDS). Czechoslovak MDS Cooperative Group.

In order to detect a possible relationship between clonal chromosomal abnormalities acquired during the course of the disease and its prognosis in patients with myelodysplastic syndrome (MDS) the authors have performed consecutive analyses in 77 patients with this disease. They were part of the large series of 209 patients cytogenetically examined during the last ten years. According to the cytogenetic findings we have distinguished three groups: 1) sixteen patients who has a normal karyotype in bone marrow cells at the beginning of the investigation and this finding remained unchanged during the course of the disease. Three of them progressed into acute leukemia (AL) without any detectable change in the chromosomal complement of the bone marrow cells; 2) twenty-five patients who had at the beginning of the study, different pathological chromosomal clones in bone marrow cells. There was no chromosomal evolution detectable during the disease; eight of them progressed into acute leukemia; 3) thirty-six patients who had either normal or pathological chromosomal findings at the first examination and in whom further clonal abnormalities had developed during the course of the disease. Twelve of them progressed into acute leukemia. Two to nine cytogenetic examinations were successfully performed with a mean of three studies per patient. The results confirmed strictly individual development of chromosomal abnormalities during the course of the disease, with an unfavorable prognosis for the patients with complex chromosomal changes. Three patients with del 7q had very poor prognosis with rapid progression of the disease. Two cases with the same acquired abnormalities (del 20q, +8, -22) transformed into acute leukemia within the period of 36 months from the onset of the disease.     

Genome analysis of Agrobacterium tumefaciens: construction of physical maps for linear and circular chromosomal DNAs, determination of copy number ratio and mapping of chromosomal virulence genes.

The phytopathogenic bacterium Agrobacterium tumefaciens is unique in that it possesses both linear and circular DNA chromosomes in addition to a plant-tumor-inducing (Ti) plasmid. We analyzed the two chromosomal DNA molecules in strain MAFF301001, whose Ti plasmid has already been sequenced completely. Physical maps of the chromosomal DNAs were constructed by Southern hybridization experiments using Pme I and Swa I fragments and short fragments bridging the Swa I fragments with special care to avoid any missing fragment. Hybridization with 16S rDNA probe showed one rDNA locus on the linear chromosome and two loci on the circular chromosome. For this bacterium to be pathogenic, not only Ti plasmid but also chromosomal genes are required. The chromosomal virulence (chv) genes (chvA, chvB, chvD, chvE, chvG, chvH, and chvI) and the chromosomal genes affecting the virulence [acvB, pgm(exoC), glgP, miaA, and ros] were successfully mapped onto 5 different regions in the chromosomal physical maps. These chv genes and the chromosomal genes affecting the virulence other than pgm and glgP were found on the circular chromosome, whereas the pgm and glgP genes were located on the linear chromosome. In contrast to the large terminal inverted repeats of Streptomyces linear chromosomal DNA, no hybridization signal was detected between left and right terminal fragments of the linear A. tumefaciens chromosome. Quantitative analysis of DNA fragments indicated that the copy numbers of the two chromosomal DNAs and the Ti plasmid are identical.     

Karyotypic evolution in Gehyra (Gekkonidae: Reptilia) IV. Chromosome change and speciation

Karyotypic data are presented for six additional species from the genus Gehyra collected in Australia, New Guinea and Fiji. C and G-banding of three of the very diverse species which all share the ancestral 2n=44 karyotype, further strengthens the phylogenetic model for the evolution of this complex. With 19 Australian species and chromosome races of Gehyra now karyotyped, it has been possible to evaluate the mode of chromosomal evolution and the role that chromosome change has played in speciation in this genus. It is clear that speciation in certain karyomorph groups has occurred allopatrically, without any gross chromosomal changes. However, in the numerous chromosome races and species which have been involved in colonizing radiations, chromosomal rearrangements have been intricately associated with the speciation process.     

Genomic integration system based on pBR322 sequences for the cyanobacterium Synechococcus sp. PCC7942: transfer of genes encoding plastocyanin and ferredoxin.

Synechococcus sp. PCC7942 recipient strains were constructed for the chromosomal integration of DNA fragments cloned in any pBR322-derived vector, which carries the ampicillin resistance (ApR) marker. The construction was based on the incorporation of specific recombination targets, the so-called 'integration platforms', into the chromosomal metF gene. These platforms consist of an incomplete bla gene (ApS) and the pBR322 ori separated from each other by a gene encoding an antibiotic (streptomycin or kanamycin) resistance (SmR or KmR). Recombination between a pBR322-derived donor plasmid and such a chromosomal platform results with high frequency in restoration of the bla gene and replacement of the chromosomal marker (SmR or KmR) by the insert of the donor plasmid. The integration into the platform depends on recombination between pBR322 ori and bla sequences only and is therefore independent of the DNA insert to be transferred. The desired recombinants are found by selection for a functional bla gene (ApR) and subsequent screening for absence of the chromosomal antibiotic marker. Gene transfer with this integration system was found to occur efficiently and reliably. Furthermore, the presence of the pBR322 ori in the platform allowed for 'plasmid rescue' of integrated sequences. The system was applied successfully for the transfer of the gene encoding plastocyanin (petE1) from Anabaena sp. PCC7937 and for the integration of an extra copy of the gene encoding ferredoxin I (petF1) from Synechococcus sp. PCC7942 itself.     

Chromosomal abnormalities and the morphology of mouse sperm heads.

In the mouse, numerous mutagens, teratogens and carcinogens have been shown to induce marked elevations in the fraction of sperm with head shape abnormalities. Since carcinogens and teratogens may act by causing genetic damage, a likely explanation of these results is that the sperm abnormalities are also caused by genetic damage. There are two more or less distinct classes of genetic damage, chromosomal aberrations and point mutations. In this paper, we provide evidence, that in general, chromosomal aberrations are not responsible for causing abnormally shaped sperm. Chromosomal aberrations could have caused abnormal sperm morphology in a number of ways. One possibility was that the mere presence of a translocated chromosome within the germ cell led to the malformation of the sperm head. A second possibility was that chromosomal imbalance, i.e., aneuploidy, duplications or deficiencies, within the spermatid or haploid cells caused abnormalities in shape. We tested these hypotheses by measuring the level of abnormally shaped sperm in mice homozygous and heterozygous for 24 various reciprocal and Robertsonian translocations. The diploid cells of these mice are known to be chromosomally balanced, containing translocated chromosomes. A predictable proportion of their gametes are, however, chromosomally unbalanced and carry translocated chromosomes. It was found that the levels of sperm abnormalities in these mice were convincingly unrelated to the levels predicted by any of the above hypotheses. Based on these results it seems that sperm abnormalities in mice are not due to the mere presence of translocated chromosomes in germ cells and also not due to chromosomal aneuploidy or duplication-deficiencies of chromosomal segments in the spermatid during development of the sperm.     

Natural transformation in Campylobacter species.

Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without the requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 10(-3) transformants per recipient cell in C. coli and 10(-4) in C. jejuni. Maximum competence was found in the early log phase of growth. Campylobacters preferentially took up their own DNA in comparison with Escherichia coli chromosomal DNA, which was taken up very poorly. Three new Campylobacter spp.-to-E. coli shuttle plasmids, which contained additional cloning sites and selectable markers, were constructed from the shuttle vector pILL550A. These plasmid DNAs were taken up by campylobacters much less efficiently than was homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 10(-4) transformants per cell were obtained. The tetM determinant, originally obtained from Streptococcus spp. and not heretofore reported in Campylobacter spp., was isolated from an E. coli plasmid and was introduced, selecting for tetracycline resistance, by natural transformation into C. coli.     

Chromosomal evidence for sibling species of the malaria vector Anopheles cruzii.

An analysis of the ovarian polytene chromosomes of Anopheles cruzii from three localities in Southeast Brazil revealed the existence of two genetic entities within this morphologically uniform taxon. These cryptic species differed in the banding patterns of the X chromosome and 3L arm. A pattern of bands that cannot be explained by the fixation of any of the known inversions in chromosome X was revealed and named chromosomal form B to distinguish it from the standard pattern of this X chromosome, form A. Each chromosomal form is characterized by a different set of inversions. The lack of heterozygotes (A/B) for these X chromosome forms in populations where both forms coexist is evidence of absence or limited gene flow between the two groups.