Chromosomal Strategies for Adaptation to Univalency

The orientation and segregation behaviour of different types of univalents, namely sex chromosomes, B chromosomes and autosomal univalents, were analysed in living spermatocytes of eight evolutionarily distant grasshopper species. The meiotic behaviour of each univalent was characterized in terms of velocity of prometaphase movements, frequency of reorientations, types of final orientation at metaphase I and modes of segregation at anaphase I. All these features were found to vary between different univalents. Certain combinations of these traits, defining a chromosomal strategy, appear commonly together in certain chromosome types, indicating that they are the result of selection acting on the chromosomes to increase transmission effectiveness. The sex univalents show in general a strategy in which all the features favouring an eventual equational segregation at anaphase I tend to be minimized. There is much more variation in behaviour among B chromosomes than among X chromosomes, which is a reflection of their heterogeneous nature. Induced autosomal univalents are studied in Locusta migratoria. They show a very irregular behaviour, indicating their lack of adaptation to univalency.     

Segregation of holocentric chromosomes at meiosis in the nematode,Caenorhabditis elegans

The meiotic segregation of the holocentric chromosomes ofCaenorhabditis elegans in both spermatogenesis and oogenesis is described. The extended kinetochore typical of the mitotic chromosome could not be differentiated on meiotic bivalents; instead microtubules appeared to project into the chromatin. The meiotic spindles formed during spermatogenesis contain centrioles and asters, while in oogenesis the spindles are acentriolar and barrel shaped. The formation of the acentriolar spindle was studied in fixed specimens by anti-tubulin immunofluorescence. Microtubule arrays were seen first to accumulate in the vicinity of the meiotic chromosomes prior to congression. At later stages, elongated spindle structures up to 13 in length were observed parallel to the surface of the embryo. Further development of the spindle appeared to involve its shortening into a barrel shape and rotation so that one spindle pole was opposed to the membrane. By anaphase the pole-to-pole spindle length reached a minimum of 3–4 . One end of each chromatid in the meiotic bivalent was labelled byin situ hybridization of a probe DNA to show that in oogenesis the chromatids were associated end-to-end in the bivalent. Furthermore, either the right or the left ends of the homologues could be held in association. At metaphase I the bivalents were oriented axially, such that kinetic activity was restricted to one end of each pair of sister chromatids. At metaphase II the chromosomes were also aligned axially.     

Direction of DNA sequences within chromatids determined using strand-specific FISH

The 5 to 3 direction of DNA strands within chromatids of metaphase chromosomes can be determined by using simultaneous hybridization of a single strand of the telomere probe and a single strand of a repetitive sequence to slides pretreated for strand-specific hybridization. The telomere probe identifies the direction of the DNA helical strand remaining in each chromatid of the metaphase chromosomes. The direction of the repetitive sequence is then determined from the direction of the strand to which it hybridizes. This method was used to determine the 5 to 3 direction of three repetitive DNA sequences, each for a different human repeat family.     

Fine structure of the XY body in the XY1Y2 trivalent of the batArtibeus lituratus

Electron microscopy of spread spermatocytes and thin sections has been used to study the sex trivalent (XY₁Y₂) of the batArtibeus lituratus. Pachytene spermatocytes in thin sections show an XY body with typical chromatin condensation that is connected to autosomal chromatin through a synaptonemal complex (SC). Microspread spermatocytes show three axes and two SC segments (a short SC and a long one) in the sex trivalent. The short paired region corresponds to synapsis between the original X and Y pieces, while the long paired region corresponds to synapsis between the Y₂ element and the homologous, autosomal piece of the compound X-chromosome. The length ratios of the three axes correspond to those of the three mitotic chromosomes, X, Y₁ and Y₂. The high packing of chromatin corresponds exclusively to the original pieces of the X and Y elements, while the autosomal regions of the X and the Y₂ axes are surrounded by autosomal-like chromatin. Thus, in this trivalent the formation of an XY body in the original sex chromosomes is not inhibited by the presence of the autosomal pieces, and typical partial synapsis between the original X and Y elements is conserved. C-banding heterochromatin seems not to be the barrier preventing the spreading of heterochromatinization towards the autosomal piece in this trivalent.     

Transforming growth factor β gene maps to human chromosome 19 long arm and to mouse chromosome 7

Transforming growth factors (TGF) are defined as biologically active polypeptides which reversibly confer the transformed phenotype onto untransformed cultured cells. They have been subdivided into two classes: type and type TGFs. TGF- acts synergistically with TGF- in inducing phenotypic transformation. TGF- can also act as negative autocrine growth factor. A human 1050-bp EcoRI cDNA fragment was used to map the human locus for TGF- by Southern blotting of DNA prepared from 17 human × Chinese hamster somatic cell hybrids. The humanspecific restriction fragments segregated with human chromosome 19 in all of 14 informative hybrids. All other human chromosomes were discordant with the TGF- bands in at least four hybrids. After in situ hybridization of the tritiated TGF- probe to normal human metaphase spreads, 151 silver grains were scored in 54 cells. Of 24 grains over chromosome 19, 16 grains (11%) lay over region 19q13.1 q13.3. Of the 54 cells analyzed, 16 (30%) had label over region 19q13.1 q13.3. Thus,TGFB is assigned to chromosome 19, subbands q13.1 q13.3. TheTgf- locus in the mouse was mapped to chromosome 7 by hybridizing a murine cDNA probe to a Chinese hamster × mouse hybrid panel. Human chromosome 19 and proximal mouse chromosome 7 share another four homologous loci.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Lichtmikroskopische Untersuchungen über die Entwicklung vonTheileria annulata (Dschunkowsky und Luhs, 1904) inHyalomma anatolicum excavatum (Koch, 1844)

Summary Fully differentiated kinetes, average length 17.6m, appeared in the haemolymph of engorged nymphs usually 17 to 20 days after repletion. Kinetes were observed at first in the salivary glands on day 18 after repletion. The kinetes then transformed into fission bodies of about 10m in diameter, mainly in type III alveoli and less frequently in type II alveoli. The fission bodies grew up to a size of about 20m after several divisions of their nucleus. At this time the ticks moulted and no further development occurred until activation. Shortly before infestation the salivary glands began to proliferate, and rapid growth of the fission bodies was observed, especially in young ticks where development of infective particles (sporozoites) was concluded within two days. Development in feeding adult ticks apparently occurred in four major steps: (1) Division of primary fission bodies (sporonts) into numerous secondary fission bodies (primary sporoblasts), (2) division of secondary fission bodies into tertiary fission bodies (secondary sporoblasts), (3) production of particles (sporozoites) by tertiary fission bodies and release of particles into the saliva, and (4) degeneration of fission bodies and their host cell but further release of particles.The host cell was stimulated to giant growth, thus its diameter increased, on average, from 15 to 110 m. Heavy infections resulting from parasitaemias of >40% caused disease and mortality in the tick population. Development was much retarded by aging. In ticks starved for six months sporozoites did not develop before day five to seven of infestation. Sporozoites may not develop at all in six to nine month old female ticks during the infestation period. The significance of the described developmental stages ofT. annulata was discussed and a sexual generation postulated. The hypothetic development ofT. annulata in its tick vector was illustrated.

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Gefördert von der Deutschen Forschungsgemeinschaft (DFG)     

Oligoclonal interspecific origin of ‘North Indian’ and ‘Chinese’ sugarcanes

Sugarcanes consist of several groups of complex polyploid forms. The origin of North Indian and Chinese sugarcanes (referred to as S. barberi and S. sinense) was investigated using genomic in-situ hybridization (GISH), detection of species-specific repeated sequences and RFLP. GISH proved their interspecific hybrid origin. Together with the distribution of species-specific repeated sequences and earlier RFLP data, the results show that both taxa are derived from interspecific hybridization between S. officinarum and S. spontaneum and that no other genus has been directly involved. RFLP indicates that the clones are clustered into a few groups, each derived from a single interspecific hybrid that has subsequently undergone a few somatic mutations. These groups correspond quite well with those already defined based on morphological characters and chromosome numbers. However, the calculated genetic similarities do not support the existence of two distinct taxa. The North Indian and Chinese sugarcanes represent a set of horticultural groups rather than established species.     

Chromosome mapping of biological pathways by fluorescence-activated cell sorting and cell fusion: Human interferon gamma receptor as a model system

Human chromosome 6 encodes both the interferon gamma receptor as well as the class I major histocompatability complex antigens, HLA-A, -B, and -C. However, the presence of chromosome 6 in somatic cell hybrids is insufficient to confer sensitivity to human interferon gamma (Hu-IFN-) as assayed by class I HLA induction; it is necessary for both human chromosomes 6 and 21 to reside in the hybrid to generate a response to Hu-IFN-. Treatment of such a hamster-human hybrid, Q72-18, with Hu-IFN- induces the class I HLA antigens. Q72-18 cells selected by fluorescence-activated cell sorting for the loss of class I HLA induction also lost human chromosome 21. Fusions of such cells to a hybrid that contains only human chromosome 21 reconstitutes HLA antigen induction by Hu-IFN-. Furthermore, fusions of hybrids containing a translocated human chromosome 6q and the HLA-B7 gene to a line containing only human chromosome 21 or a translocated 21q also reconstitutes HLA-B7 mRNA and antigen induction by Hu-IFN-. Thus the segregation of cells on the basis of a biological effect by fluorescence-activated cell sorting and reconstitution by hybrid fusion provides a strategy by which some biological pathways can be mapped at a chromosomal level.     

Chromatin structure of active and inactive human X-linked phosphoglycerate kinase gene

We have carried out a comparative analysis of DNase I sensitivity of the active and inactive X-linked phosphoglycerate kinase (Pgk)genes in human lymphoblast and fibroblast cultured cells. Three DNase I-sensitive regions were detected: a 5 hypersensitive site, a sensitive region in the interior of the gene and a 3 slightly sensitive site which we previously reported and have now mapped with some precision. A comparison of these sensitive sites in single and multiple X cell lines indicates that the sensitive sites are unique to the active X chromosome. A similar study of an X-linked Pgkpseudogene shows no difference in DNase I sensitivity between the pseudogenes on the active and inactive X chromosomes. These latter results imply that sex chromatin does not confer a unique level of DNase I resistance to DNA on the inactive X chromosome. The exact role of sex chromatin in differential DNase I sensitivity of genes on the inactive and active X chromosomes is discussed.     

Regional localization of aβ-galactosidase locus on human chromosome 22

Human white blood cells with an X/22 translocation [46, XX, t(X; 22)(q23; q13)] were fused with Chinese hamster cells. The isolated hybrids were analyzed for human chromosomes and 21 enzyme markers. An electrophoretic technique for studying the -galactosidase isoenzymes in man-Chinese hamster hybrid cells was developed. Immunological studies showed that the -galactosidase marker studied in these hybrids did contain immunological determinants of human origin. Furthermore the results provided evidence that a locus for -galactosidase is situated on chromosome 22 distal to the breakpoint in q13.     

Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.)

Procedures for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) have been developed for chickpea (Cicer arietinum). Suspensions of intact chromosomes were prepared from root tips treated to achieve a high degree of metaphase synchrony. The optimal protocol consisted of a treatment of roots with 2mmol/L hydroxyurea for 18h, a 4.5-h recovery in hydroxyurea-free medium, 2h incubation with 10µmol/L oryzalin, and ice-water treatment overnight. This procedure resulted in an average metaphase index of 47%. Synchronized root tips were fixed in 2% formaldehyde for 20min, and chromosome suspensions prepared by mechanical homogenization of fixed root tips. More than 4×10⁵ morphologically intact chromosomes could be isolated from 15 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing eight peaks, representing individual chromosomes and/or groups of chromosomes with a similar relative DNA content. Five peaks could be assigned to individual chromosomes (A, B, C, G, H). The purity of sorted chromosome fractions was high, and chromosomes B and H could be sorted with 100% purity. PCR on flow-sorted chromosome fractions with primers for sequence-tagged microsatellite site (STMS) markers permitted assignment of the genetic linkage group LG8 to the smallest chickpea chromosome H. This study extends the number of legume species for which flow cytogenetics is available, and demonstrates the potential of flow cytogenetics for genome mapping in chickpea.     

Completion of the Four Large Gene Sequences of Porcine Group C Cowden Rotavirus

The terminal nucleotide sequences of group C Cowden rotavirus gene segments 1-4 were determined. When compared with the published sequences, we found 14 to 29 additional nt at the 5 ends of the four reported gene sequences. For the 3 ends, we observed an additional 16 nt in gene 2 and 14 fewer nt in gene 4.     

γδ T Lymphocytosis Associated with Common Variable Immunodeficiency1

We present the case of a 28-year-old Caucasian female with common variable immunodeficiency (CVID) since age 5 who had a long history of hospitalizations for unexplained fevers and pulmonary infiltrates. The patient developed mild lymphocytosis 7 months prior to our evaluation. Flow cytometry of peripheral blood revealed an expansion of T lymphocytes, mild CD4 T lymphocytopenia, and a reduced CD4/CD8 ratio (0.2). Two subpopulations of T lymphocytes were found (CD3⁺/CD4^–/CD8⁺, 47%; CD3⁺/CD4^–/CD8^–, 53%), the vast majority of which expressed V-1. An infectious cause for the patient's T lymphocytosis could not be found. The sputum was chronically colonized with Staphylococcus aureus, and the organism produced TSST-1 in vitro. A bronchoalveolar lavage (BAL) revealed marked lymphocytosis, but T lymphocytes were not overrepresented in the BAL. Lymphocyte functional studies revealed poor proliferative responses to mitogens and staphylococcal superantigens and diminished cytokine production. V-1 T lymphocytes from the patient's blood were not expanded in vitro in response to staphylococcal superantigens. TCR gene rearrangement studies confirmed the presence of J and J1 clonal rearrangements accounting for only a small subpopulation of the T lymphocytes. These studies were repeated 5 months later and were unchanged. A bone marrow biopsy was negative for leukemia. Hence, the cause of the patient's T lymphocytosis could not be determined despite evaluation for underlying malignancy, occult infection, or superantigen-driven stimulation. The patient ultimately died of progressive respiratory insufficiency. The state of current knowledge regarding T lymphocytosis, decreased production of T lymphocytes, and a low CD4/CD8 ratio in association with CVID is discussed.     

Regional mapping of RBP4 to 10q23→q24 and RBP1 to 3q21→q22 in man

The human gene coding for RBP4 has been assigned to 10q2324 using a panel of somatic cell hybrids and in situ hybridization experiments. The mapping of the human RBP1, previously assigned by our group to chromosome 3 using a panel of somatic cell hybrids, was restricted to the region 3q2122 using in situ experiments and Southern blots of genomic DNA from a hybrid retaining a portion of chromosome 3.R.F. is recipient of a Research Grant from A.I.R.C.     

Three distinct loci on human chromosome 21 contribute to interferon-α/β responsiveness

The species specificity of interferons (IFNs) depends on restricted recognition of these ligands by multisubunit cell surface receptors. Expression of the human receptor subunit IFNAR in mouse cells conferred sensitivity only to one subtype of human IFN, IFN-B. Other genes on human chromosome 21 were required for responses to other subtypes of type I IFN. In contrast, IFNAR expression in hamster cells did not confer sensitivity to any human IFN tested, including IFN-B. Using human-hamster somatic cell hybrids, we mapped theIfnabr gene, encoding a ligand-binding subunit of the IFN-/ (type I) receptor, to human chromosome 21.Ifnabr colocalized withIfnar to the distal region of q22.1. The presence of a chromosomal fragment encoding IFNABR and IFNAR was also not sufficient to confer sensitivity to human IFN. In contrast, hybrids carrying in addition the region 21q22.2 showed a full response to human IFN-B, suggesting that a gene located in this region encodes a third factor required for type I IFN receptor activity.     

Interferon-β-related DNA on human chromosome 4

A DNA subclone (pPE-4000) derived from the B4 interferon--related human genomic DNA clone was used as a probe in blot-hybridization experiments of DNA from a panel of human-rodent somatic cell hybrids containing overlapping subsets of human chromosomes. The DNA hybridization experiments showed that the B4 IFN- locus is localized to human chromosome 4. A provisional regional assignment to 4q12-qter was also obtained. Thus available hybridization data implicate human chromosomes 2, 4, and 9 in the human IFN- system while the available biological data also implicated human chromosome 5.     

Genes for C4b-binding protein α- and β-chains (C4BPA andC4BPB) are located on chromosome 1, band 1q32, in humans and on chromosome 13 in rats

C4b-binding protein is involved in the regulation of the complement system. It is a multimeric protein composed of seven identical -chains and a single copy of a unique -chain. The latter was identified only recently and its structure determined by cDNA cloning. Both subunits in C4b-binding protein belong to the same superfamily of proteins composed predominantly of tandemly arranged short consensus repeats (SCR) approximately 60 amino acid residues in length. The gene for the human -chain is known to be located in a gene cluster on chromosome 1, band 1q32, which is called the regulators of complement activation (RCA) gene cluster. We have used cDNA probes for both - and -chains of human C4b-binding protein to localize their genes with an in situ hybridization technique. We find the genes for both chains to be located on chromosome 1, band 1q32, in the human. This suggests that the -chain gene is also a member of the RCA gene cluster and that the - and -chain genes are located close to each other. The cDNA probes for the - and -chains also were used to screen mouse-rat somatic cell hybrids using Southern blotting to localize their genes in the rat. Both the - and -chain genes were shown to be located on chromosome 13 in the rat. These are the second and third genes to be located on rat chromosome 13, and the results suggest that the genes for the - and the -chains together with the gene for coagulation factor V represent a conserved chromosomal region in rat and man.     

Recognition by peptide mapping of three different structural groups of outer membrane protein YOP-1 of Yersinia enterocolitica and Yersinia pseudotuberculosis

The structural relation of YOP-1 of european and american Yersinia enterocolitica serotypes O3, O9, O5, 27, and O8 and O20, respectively, and Y. pseudotuberculosis serotypes I, II, and III was compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping using Staphylococcus aureus protease V8. Apparent molecular weights of YOP-1 ranged from 206,000 (O3) to approx. 180,000 (O8). According to their respective peptide maps YOP-1 of the european and american Y. enterocolitica serotypes and Y. pseudotuberculosis serotypes could be assigned to three different groups. Evaluation of several isolates of Y. enterocolitica serotypes O3, O9, and O8 by peptide mapping indicated that YOP-1 is conserved within a serotype. However, one serotype O8 isolate differed from the consensus peptide pattern of the other serotype O8 and O20 isolates. The similarity of the peptide patterns of Yersinia serotypes which predominate in certain geographical locations, i. e., european and american Y. enterocolitica serotypes, suggest common evolution of YOP-1 of these serotypes independent of the evolution of the other serotypes.