Further studies on polyploid amphibians (Ceratophrydidawe)

Odontophrynus cultripes Reinhardt and Lutken, 1862 has 22 chromosomes in its diploid complement. Spermatocyte I contained 11 ring bivalents and metaphase II exhibited 11 chromosomes. Odontophrynus americanus (Duméril and Bibron) 1882 has 44 chromosomes in somatic as well as germ cells, these can be sorted into 11 groups of homologues. Metaphase I showed varying numbers of quadrivalents and metaphase II exhibited 22 dyads. Ceratophrys dorsata Wied., 1824 has 104 chromosomes in somatic and germ cells; these 104 chromosomes comprise 8 each of 13 kinds of homologues. The spermatocyte I contained ring octovalents and other multivalents, and metaphase II 52 chromosomes. The above findings indicate that evolution by polyploidization occurred in South American frogs belonging to the family Ceratophrydidae.This work was supported by a grant (GM-14577-01) from the National Institute of General Medical Sciences U. S. Public Health Service.     

Metaphase synchronization and chromosome preparation from the OK opossum cell line having a potentially isolatable X chromosome

Summary As part of a study on X chromosomes, metaphase cell synchrony and chromosome isolation methods were developed for the opossum (Didelphis virginiana) kidney epithelial cell line (OK). The cell synchrony yielded large amounts of metaphase cells using a relatively simple method in which a key feature was a calcium- and magnesium-free balanced salt wash. A neutral pH chromosome isolation method was developed for the kidney epithelial cells, because they were somewhat difficult to disrupt fully by other methods. FACS IV flow microfluorometric analysis of OK chromosomes confirms a clear difference between the sizes of opossum X chromosomes and autosomes.     

Chromosomal effects on peptidase activities inDrosophila melanogaster

The peptidase system inDrosophila melanogaster (dipeptidase-A, -B, and -C and leucine aminopeptidases G and P) was used as a model to study the effects of modifier genes on activity of enzymes with similar functions. A screen of X, second, and third chromosome substitution isogenic lines revealed the presence of activity modifiers for peptidases on all three chromosomes. Correlation analyses indicated that covariation between some of the peptidase activities is independent of genetic background, while others are associated with variable second chromosomes. Chromosome-specific effects onK _m ,V _max, and specific activity of partially purified peptidases were also detected. Moreover, a repeatable technique using anion-exchange column chromatography allowed the characterization of possibly two putative peptidic enzymes, glycyl-l-isoleucine-ase andl-leucyl-l-proline-ase, whose kinetic properties differ from the dipeptidases and the leucine aminopeptidases. These findings confirm the existence of activity modifiers for peptidases, much like other enzymes inDrosophila melanogaster. These studies were supported by grants from the National Institutes of Health (GM42-115-01A1), the Whitaker Foundation of the Research Corporation (C-2560), and the National Science Foundation (USE 8951018) to Kazuo Hiraizumi.     

Chromosomes of the murine leukemia virus indicator cell line XC

A cell line derived from the Rous Sarcoma Virus induced rat tumor XC (Svoboda), which was recently utilized as an indicator for the presence of murine leukemia virus growing in mouse cells, has been examined karyologically. The cells differ considerably from each other as well as from the normal rat karyotype (Rattus norvegicus, 2n=42). The modal chromosome number is 41. All cells bear one or more chromosome markers in common as well as non-rat-like chromosomes, but rat-like chromosomes still preserve the identity of species origin.Supported by Contract No. PH 43-63-13 between the University of California and the National Cancer Institute, National Institutes of Health (Special Virus Cancer Program).     

On the karyotype of the tahr Hemitragus jemlahicus and the Y-chromosome of goats and sheep

Somatic chromosomes of a female and male Himalayan thar, Hemitragus jemlahicus (H. Smith) are described. The diploid number is 48, there are 12 atelocentric and 34 telocentric autosomes in both sexes, the X-chromosome is meta- or submetacentric. The morphological appearance of the Y-chromosome is compared with that of other bovid species including recent observations on the goat Capra hircus.Supported by Contract No. PH 43-63-13 between the National Cancer Institute of the National Institutes of Health and the University of California.     

The elimination and differentiation of chromosomes in the germ line of sciara

The germ line chromosomes of S. coprophila have been followed from the time of origin of the germ cells up to the time of meiosis in the male and up to first larval molt in the female. The mechanism which prevents the accumulation of L (limited) chromosomes in the germ line is a unique process of chromosome elimination: it occurs in male and female embryos after the germ cells have migrated from the pole plasm to the definitive gonad site, and it involves the movement of whole L chromosomes through the nuclear membrane into the cytoplasm. The extra paternal X chromosome is eliminated from the germ cells at the same time and in the same manner. Following this elimination there is a cytological differentiation of the chromosomes remaining inside the nucleus. First, the 4 paternal homologues of the regular complement undergo a loosening of coils and become light-staining whereas the maternal homologues remain condensed like the L's. Next, the L chromosomes undergo a process of extreme attenuation and dispersion following which they return to the condensed state. H³-thymidine autoradiography on gonial and premeiotic cells in the testis reveals that the L chromosomes undergo DNA replication at the end of the S period, also that there are asynchronies in DNA synthesis among the regular chromosomes. The phenomena of differential chromosome staining and asynchronous DNA replication are considered in the light of current theory regarding heterochromatization and gene inactivation, also in relation to the phenomenon of chromosome imprinting encountered in this genus.The studies reported here were supported by the National Science Foundation grants GB-42 and GB-2857, and in part by Contract No. AT-(40-1)-2690 under the Division of Biology and Medicine, U.S. Atomic Energy Commission.Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Department of Botany, Columbia University. This work was carried out in the laboratory of Professor J. Herbert Taylor and has been supported in part by U.S. Public Health Training Grant No. 2 T 1-GM-216-05. Grateful acknowledgement is made to Professor Spencer W. Brown, Department of Genetics, University of California, Berkeley, in whose laboratory the final studies were completed.     

Chromosome studies of the cultured cells of two species of side-necked turtles (Podocnemis unifilis and P. expansa)

The diploid chromosome number of two species of sidenecked turtles (Podocnemis unifilis and P. expansa) was found to be 28. Under normal culture conditions, half of the chromosomes of P. unifilis consistently show one or two clear secondary constrictions. In P. expansa, the incidence of cells with chromosomes bearing secondary constrictions and the number of such chromosomes per cell are less. Cells of two P. unifilis cell lines maintained a normal diploid karyotype for two years following their initiation. Then one cell line shifted to a hypodiploid mode of 27 and half of the population of the second line became pseudodiploid, the other half remaining diploid. A single six-month-old cell line from P. expansa has maintained a normal diploid mode through 10 passages.Supported in part by grant-CA 08737 from the National Cancer Institute.     

Methylation status of ribosomal RNA gene clusters in the flow-sorted human acrocentric chromosomes

Southern blot analysis of the human acrocentric chromosomes that were flow-sorted from B-lymphoblastoid cell line GM130B revealed that the sensitivity of the ribosomal RNA (rDNA) gene clusters to the restriction enzyme NotI differs among these rDNA-containing chromosomes: the rDNA clusters of Chromosomes (Chr) 13, 14, and 15 are much more sensitive to NotI digestion than those of Chrs 21 and 22 in this particular cell line. Detailed analysis by use of methylation-sensitive enzymes HpaII and HhaI and methylation-insensitive enzyme MspI confirmed the significant variation in the methylation status of rDNA clusters among these chromosomes. Quantitative analysis by fluorescent in situ hybridization (FISH) indicated that copy number of rDNA varies among individual chromosomes, but the average copy number in the acrocentric Chrs 21 and 22 is significantly greater than that of the Chrs 13, 14, and 15 in GM130B cells. Similar analysis reveals that the methylation status of rDNA clusters in another B-lymphoblastoid cell line GM131 was different from that of GM130B. These data together indicate that the copy number and methylation patterns of rDNA clusters differ among individual acrocentric chromosomes in a given cell line, and they are different among cell lines.     

The fine structure of the kinetochore in meiotic cells of Tradescantia

Summary Observations on Tradescantia cells in the second meiotic division revealed distinct regions in meiotic chromosomes. These areas 1) consistently stain less dense than the chromosomes themselves, 2) have direct connections with the chromosomes at certain points, and 3) serve as focal points for spindle tubules during meiosis. These lighter staining regions are similar in character to kinetochores (centromeres) found in animal cells.

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Zusammenfassung Es wurde beobachtet, daß bestimmte Regionen der Chromosomen von Tradescantia zu der zweiten meiotischen Teilung 1. durchgängig sich weniger stark färben als die Chromosomen selbst, 2. daß sie an gewissen Punkten direkte Verbindungen mit den Chromosomen haben, und 3. daß sie während der Meiosis als Sammelpunkte für Spindeltubuli dienen. Diese schwächer gefärbten Stellen werden als Kinetochoren (Centromeren) interpretiert.

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This work was supported by grant GB-3330 from the National Science Foundation to Dr. Wayne Ferris, and the training grants GM 00441 and DE 00184 from the National Institutes of Health.     

The liver of the slender salamander Batrachoseps attenuatus

Summary The structure of the crystalline inclusions found in Batrachoseps liver cells is described and it is shown that the most symmetric unit cell upon which the crystal lattice is built is a face-centered cube. Taking into consideration the physical properties of a face-centered cubic structure, an attempt is made to determine the nature of the macromolecules that comprise the crystal. It is concluded on the basis of available evidence that the macromolecules probably represent serum lipoproteins. The intracellular synthesis of the crystals and the possible functions they may subserve in the animal are discussed. A comparison is made between the crystals and granules in rat hepatocytes discussed by Bruni and Porter (1965).Research supported by grant RG-6729 from the Institute of General Medical Sciences, National Institutes of Health, United States Public Health Service.     

Purine nucleotide synthesis in lymphoblasts cultured from normal subjects and a patient with Lesch-Nyhan syndrome

Human lymphoblasts derived from normal and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient individuals have been maintained in permanent tissue culture, and comparative studies of their purine metabolism have been undertaken. In agreement with previous observations in fibroblasts, the HGPRT-deficient lymphoblasts (less than 2% normal HGPRT activity) demonstrate threefold increases in the production of purines by the de novo pathway and four- to eightfold increases in intracellular concentrations of 5-phosphoribosyl 1-pyrophosphate (PRPP). The activities of the enzymes of purine metabolism responsible for production and utilization of PRPP were measured under optimal conditions in each cell line. The activities of adenine phosphoribosyltransferase (APRT), PRPP synthetase, and PRPP amidotransferase were independent of cell density and were not significantly different in the two cell lines. The K _m values of the common substrate, PRPP, were determined in normal lymphoblast extracts for APRT (K _m of 0.033 mM), HGPRT (K _m of 0.074 mM), and PRPP amidotransferase (K _m of 0.3 m M). The relatively low affinity of PRPP amidotransferase for PRPP suggests that deficiency of the HGPRT enzyme with its attendant increase in PRPP concentration should be accompanied by increased in vivo activity of PRPP amidotransferase, the first and presumed rate-limiting enzyme of de novo purine biosynthesis.This work was supported in part by National Institutes of Health Grants AM-05646, AM-13622, and GM-17702.     

Development,characterization, and viral susceptibility of a feline (Felis catus) renal cell line (CRFK)

Summary Cell line CRFK, derived from kidney tissue of a normal domestic kitten, was initiated in 1964. With intermittent periods of storage in the frozen state, it has been grown in vitro during more than 200 passages, without apparent loss of susceptibility to selected viruses. Various herpesviruses and feline viruses belonging to differnet virus groups grow readily and with distinct, cytopathic features. The cells now grow as a smooth monolayer of epithelial-like cells; most have 37 chromosomes (2n−1) and are thus aneuploid for cat karyotype. Three distinct marker chromosomes are identified. The cell line, which is free of mycoplasmal contaimination, is useful in feline virus research and diagnostic medicine and has become of particular interest in cancer research. Supported in part by Contract E73-2001-NO1-CP-3-3237 within the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, Public Health Service and the Morris Animal Foundation and General Research Support funds of the New York State Veterinary College. CRFK cells from stock provided by C. G. Fabricant are available for distribution to investigators from the Cell Culture Laboratory, Naval Biomedical Research Laboratory.     

Trisomy associated with loss of maturation capacity in a long-term embryogenic culture of Abies alba

 Karyological studies were made on a 6-year-old embryogenic cell line of Abies alba. Embryogenic cells were obtained from a mature zygotic embryo cultivated on modified MCM-medium and subcultured every 3 weeks. Three years after induction, part of the cell line was transferred to media supplemented with 500 or 1000 mg l^-1 caseine hydrolysate and 500 mg l^-1 L-glutamine. Approximately 3 years after addition of the organic nitrogen to the medium, morphological changes such as malformation of the suspensor cells and a loss of maturation capacity occurred. Chromosome counts revealed that all cells cultivated on medium with organic nitrogen were trisomic. Fluorescent-banding methods and comparison with an euploid cell line showed that the additional chromosome belonged to the group of long, metacentric chromosomes of Abies alba without secondary constriction. Those cells cultured on medium not supplemented with caseine hydrolysate and L-glutamine retained a stable chromosome number of 2n=24. Both normal and deformed suspensor cells were observed. The maturation frequency was very low. The emergence of aneuploidy within one cell line could be the consequence of high selection pressure caused by the different culture conditions. Received: 27 February 1997 / Accepted: 7 March 1997     

On the cytotaxonomy of phasmids (Phasmatodea)

Summary A wide diversity in chromosome complement is found in two species of phasmids of the primitive group Prisopini—Prisopus ariadne Hebard and Prisopus berosus Westwood. P. ariadne has a diploid male complement of 28, comprising 13 pairs of relatively large mediokinetic autosomes and Neo XY sex chromosomes. P. berosus, 2n =49, has relatively small autosomes most of which are mediokinetic, and retains the XO—XX sex mechanism. Chromosomal polymorphism in this species is suggested by the presence of an unequal pair of autosomes and a structural differentiation in the X in one of two males studied.The relative amount of DNA per nucleus in male germ cells (Peulgen cytophotometry) shows a significant difference in total chromosomal content between the complements of the two species.These data are discussed with reference to the cytotaxonomy of phasmids.Supported in part by research grant G-4370 from the National Institutes of Health, Public Health Service.     

Biosynthesis,processing, and extracellular release of α-l-fucosidase in lymphoid cell lines of different genetic origins

In humans, the quantity of α-l-fucosidase in serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. Lymphoid cell lines derived from individuals with either low, intermediate, or high α-l-fucosidase in serum were established. Steady-state levels of intracellular and extracellular α-l-fucosidase as well as rates of synthesis and secretion of enzyme overlapped among the cell lines. Thus,_vivo} serum phenotypes were not expressed in this system. No appreciable differences in the qualitative processing of newly made α-l-fucosidase were observed among these lymphoid cell lines. Cells pulse-labeled with³⁵S-methionine from 0.25 to 2 hr had an intracellular form of enzyme with aM _r=58,000. Cells pulsed for 1.5 hr and chased for 21 hr with unlabeled methionine had an intracellular form ofM _r=60,000 and an extracellular form ofM _r=62,000. All three enzyme forms were glycoproteins with a common polypeptide chain ofM _r=52,000 but with different carbohydrate moieties. No evidence for a high molecular mass precursor form of α-l-fucosidase was found. Fucosidosis is a rare, inherited disease in which α-l-fucosidase activity in tissues and body fluids is low or absent. The mutations for fucosidosis and the serum polymorphism map separately. Lymphoid cells from two siblings with fucosidosis had 8-fold to 341-fold less intracellular α-l-fucosidase protein with 11-fold to 56-fold lower specific activities than control cells. Residual mutant enzyme was a glycoprotein with a polypeptide chain virtually the same size (M _r=52,000) as control enzyme. However, residual mutant enzyme was hypoglycosylated and hypersecreted as compared to control enzyme. This research was supported by National Institutes of Health Grant DK 32161.     

Autosomal polymorphism due to pericentric inversions in the deer mouse (peromyscus maniculatus), and some evidence of somatic segregation

The existence of extensive autosomal polymorphism due to pericentric inversions in a colony of deer mice, Peromyscus maniculatus, has been reported (Arakaki and Sparkes, personal communication). A background of intersubspecific hybridization was suspected to have contributed to the observed polymorphism. The present study describes findings on two separate subspecies, P. m. hollisteri and P. m. bairdii. Autosomal polymorphism existed within each subspecies. In P. m. bairdii, the two polymorphic autosomal pairs were identified. In one pair, a large subterminal element was homologous to an acrocentric of comparable size, while in the other pair, a small mediocentric and an acrocentric were homologous. Among splenic cells of animals made heteromorphic for the larger of the two autosomal pairs, the tendency toward reconstitution of homomorphic cell types was observed. This was taken as evidence of somatic segregation among immunologically competent cells.Dedicated to Professor J. Seiler on the occasion of his 80th birthday.This work was supported in part by grant CA-05138 from the National Cancer Institute, U. S. Public Health Service. Contribution Number 53-65, Department of Biology, City of Hope Medical Center.     

Supernumerary chromosomes in the human bed bug,Cimex lectularius Linn. (Cimicidae:Hemiptera)

The chromosome complement in the human bed bug, Cimex lectularius Linn., is 26+X₁X₂Y in the male and 26+X₁X₁X₂X₂ in the female. However, a population from Cairo, Egypt has 4 supernumerary X chromosomes. In the hybrid between the Berkeley population (with no supernumeraries) and the Cairo population (with 4 supernumeraries), the behavior of supernumeraries was observed during embryogenesis and oogenesis as well as spermatogenesis.In embryogenesis the transmission of supernumeraries was quite regular. However, one chromosome may sometimes be eliminated early in the germ line. This abnormality could induce the variations in chromosome number encountered in later stages. In the first meiotic division, some of the supernumeraries were nondisjunctional. Moreover, in the second division, some supernumeraries were eliminated. These results show that there is a tendency towards a decrease in the number of supernumeraries in the hybrids.Although the supernumeraries behave like X chromosomes, they seem not to be important for sex determination and appear to be largely or entirely inert genetically. Supernumeraries in the bed bug originate from small fragments caused by structural rearrangement. They are increased by an accumulation mechanism. Supernumeraries in the bed bug appear to be of relatively recent origin. The phylogenetic sequence in their development was probably from none to a stabilized number of four in the Old World. Then the supernumeraries were lost in two specialized lines, Cimex columbarius Jenyns on domestic birds in Europe and Cimex lectularius Linn. on man in the Western Hemisphere.This study was carried out under U.S. Public Health Service Grant (GM-13197).     

An established spleen cell line fromBairdiella chrysura

Summary A cell line, SP-2, was established from spleen tissue ofBairdiella chrysura (the silver perch). The line is susceptible to lymphocystis virus and the amphibian LT-1 virus but refractory to six additional viruses. The modal chromosome number of primary silver perch cells is 48, but SP-2 cells are heteroploid. For growth, Leibovitz L-15 medium supplemented with fetal bovine serum and sodium chloride (to 0.150m) was employed. Cells replicated best at 25° to 28° C. Funds for this investigation were supplied by the National Oceanographic and Atmospheric Administration, Office of Sea Grant No. 04-3-158-58.     

Establishment of a galactocerebrosidase-deficient twitcher mouse cell line that expresses galactocerebrosidase activity in hybrids with control human fibroblasts

Summary Primary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation of skin obtained from 3-d-old littermates of atwi+/twi×twi+/twi mating. Galactocerebrosidase activity remained deficient for two twitcher cell lines, TM-1 and TM-2, and both lines demonstrated an initial period of growth decline, followed by accelerated growth. The TM-2 line has been subcultured for more than 3.5 yr, has a modal chromosome number of 63, a doubling time of approximately 16 h, and has remained galactocerebrosidase deficient throughout its life span. These data indicate this to be an established twitcher cell line that can be continuously maintained in culture as a transformed galactocerebrosidase-deficient mouse cell line. This established line was rendered 6-thioguanine resistant so that the cells could be fused with control human fibroblasts and selected for hybrid lines in hypoxanthine-aminopterin-thymidine medium. Also, the established twitcher cells were crossed with neomycin-resistant control human fibroblasts and selected in G418 medium. Several of the hybrid lines from both crosses had higher than deficient levels of galactocerebrosidase activity initially, followed by a decrease to twitcher levels during subculture, whereas other lines retained high levels of activity. These results indicate that twitcher-human somatic cell hybrids will express galactocerebrosidase activity and thus may be useful for determining the human chromosome or chromosomes associated with this expression. Partial support for these studies was provided by a National Institutes of Health AREA grant (HD21222-01) and a NIH subcontract to Clark University from the Shriver Center for Mental Retardation This research forms a portion of studies performed to fulfill the requirements for the Ph.D. degree in biology at Clark University for J. T. K.     

Metaphase synchronization and chromosome preparation from the OK opossum cell line having a potentially isolatable X chromosome

As part of a study on X chromosomes, metaphase cell synchrony and chromosome isolation methods were developed for the opossum (Didelphis virginiana) kidney epithelial cell line (OK). The cell synchrony yielded large amounts of metaphase cells using a relatively simple method in which a key feature was a calcium- and magnesium-free balanced salt wash. A neutral pH chromosome isolation method was developed for the kidney epithelial cells, because they were somewhat difficult to disrupt fully by other methods. FACS IV flow microfluorometric analysis of OK chromosomes confirms a clear difference between the sizes of opossum X chromosomes and autosomes.