Chromosomal changes in eight cell strains of the tasmanian rat-kangaroo,Potorous tridactylis (Marsupialia). Male and female heart fibroblasts cultured in vitro

Eight cell strains, derived from the hearts of a male and a female Motorous, were followed during their in vitro cultivation.All three male cell strains started as normal diploid cell strains. One of them, 2Hpo stayed diploid until passage 59, when the cells were frozen and stored at –96°C. After a period of growth retardation, that lasted two months, 1Hpo showed aneuploidy, the cells having 22–24 chromosomes. The cells with 23 chromosomes formed about 30% of the population. These cells predominantly missed the chromosomes 2, 3 and Y₁, from the tetraploid set. In the other cells no consistent pattern was observed. The cell strain 4Hpo did not show aneuploidy after three months of growth retardation. At the last passage (nr. 24) before death, it showed 25% diploid cells and 40% tetraploid cells.Three female strains were initiated on fibrin clot, two on plasm clot. No differences in growth and chromosomal changes, due to the different embedding media, were observed. All the strains started as diploid (2n=12) cell strains. The chromosomal changes that occurred showed many differences. Three cell strains (5Hf, 7Hp, 52Hf) died without showing any pattern in the aneuploid cells. One cell strain (53Hf) showed an aneuploid cell population with a stemline of 14 chromosomes. The cell strain (8Hp) showed different changes in ploidy. After 50 passages, it changed from diploid to aneuploid (19 chromosomes per cell in the stemline). Twenty passages later diploid cells started to dominate the population again (80% at passage 85). Then a new aneuploid population with a stemline of 18 chromosomes (30% triploid cells) arose, and the strain survived as a permanent line.The work was carried out, in part, under the association between Euratom and the University of Leiden, contract Nr. 052-64-I BIAN, and it also received support form the Foundation for Basic Medical Research (FUNGO).     

Chromosomal changes in two strains of human fibroblasts cultured in vitro

The chromosomal changes that occurred in two freshly initiated human cell strains, during their in vitro life were followed. Both strains are characterized by slow growth, and both show a period of delayed growth. In one of the strains the chromosomal composition showed changes just before the period of slow growth. After this period both cell strains showed several kinds of aneuploid chromosome complements, mainly in the hypotetraploid region. Aberrant chromosomes were present at the end of the in vitro life of both strains.The work in this paper was supported by the Foundation for Basic Medical Research (FUNGO), and the Association between Euratom and the University of Leiden, contract No. 052-64-1-BIAN.     

Comparative lactation in two species of rat-kangaroo (Marsupialia)

1. Milk composition and energy content was measured in two related marsupials, Bettongia gaimardi and Potorous tridactylus. 2. Although protein content was consistently higher in bettong milk, lactational trends in both species are similar to those reported for other marsupials. 3. The faster relative growth rate of the bettong may be correlated with the increased protein and a higher milk intake.     

The distribution of x-ray induced chromosomal abnormalities in rat kangaroo (Potorous tridactylis) cells in vitro

Synchronized cells of Potorous tridactylis (female origin) were irradiated with X-rays in vitro at different times during the cell cycle. Chromosome aberrations were scored in the first metaphase after irradiation.It is shown that the distribution of breaks and gaps in the chromosome arms is different at different cell-cycle times, and is non-random.     

Characteristics of quantitative karyotypic variability in cell line of kidney from rat kangaroo (Potorous tridactylis)

The goal of this work was to investigate the numeral karyotypic variability in different sublines (MT, M2). These sublines are formed spontaneously from the main cell line (M) and have modal number of chromosomes 9 and 10, MSVK (main structural variant karyotype)--3 + 3 + 1 + 2 and 3 + 4 + 2 + 1. There are general regulations which were originally got for the line M. In particular: 1) nonrandom character of cell distribution according to the number of chromosomal deviations from MSVK; 2) specific character of deviations of each chromosome from MSVK; 3) presence of significant connections between separate chromosomes by simultaneous, mainly single directed numeral deviations. These three lines (M, MT, M2) were compared and some differences were found: 1) different frequencies of deviations from MSVK; 2) the same chromosomes have tendency to different numeral deviation; 3) the specificity of some significant connections between separate chromosomes by simultaneous numeral deviations. These results lead us to a conclusion that the balance of numerical karyotypic structure in cell populations depends on the regulations connected with the character of deviations according to the number of chromosomes from MSVK which has the largest selected advantage. Each line has its own specific limits of karyotypic variability.     

Ultrastructure of the mature spermatozoon of the musky rat-kangaroo, Hypsiprymnodon moschatus (Potoroidae: Marsupialia)

It is currently accepted that Hypsiprymnodon moschatus is a basal macropod, retaining several primitive features from the ancestral phalangeroid that gave rise both to modern possums and macropods. Sperm ultrastructure is frequently found to provide informative characters for phylogenetic analysis as these features are not strongly selected for and are thus unlikely to be confounded by effects such as convergence. Caudal epididymal biopsies were taken from two male H. moschatus and prepared for transmission and scanning electron microscopy in order to study mature spermatozoan ultrastructure. Within the diprotodont group, several features were found to be unique to H. moschatus. These were an unusual acrosome covering nearly 100% of the dorsal nuclear surface, a midpiece fibre network which is loose, indistinct and extends to the anterior‐most aspect of the midpiece, a nucleus that is very streamlined, while the principal piece is comparatively short, and a mitochondrial helix and annulus which are similar to those of dasyurids. Also reported is the presence of a fibrous network in the connecting piece, not previously reported for any marsupial.     

Precopulatory and mating behaviour in the tasmanian bettongBettongia gaimardi desmarest (Marsupialia: Potoroidae)

Intersexual interactions in Tasmanian bettongs (Bettongia gaimardi) were observed in dyadic combinations. Several behaviours were associated with agonistic confrontations and occurred in predictable response sequences. There was no evidence of a consort period between males and pre-oestrous females. Overt sexual interactions occurred only on the night of oestrus, coincidentally with the maintenance of close spatial proximity. During copulation, multiple intromissions were regularly achieved but the intervals between successive bouts of these acts generally increased. Males were also observed to engage in regular attempts at social (urogenital) investigation and were apparently able to detect the reproductive state of all resident females, presumably utilising olfactory cues. The brevity of sexual interactions occurring between individuals contrasts with the more complex repertoire of the gregarious large macropodoid marsupials.     

Changes in milk composition during lactation in the potoroo, Potorous tridactylus (Marsupialia: Potoroinae)

Milk samples from captive potoroos were analysed for composition during weeks 3-25 of the lactation period. During pouch residence, up to week 16, carbohydrate levels were high, ranging from 9 g 100 ml-1 at week 5 to 15 g 100 ml-1 at week 15; fat levels were consistently low, at around 2 g 100 ml-1; protein levels gradually increased from 5 g 100 ml-1 before week 10 to 12 g 100 ml-1 at week 16. Growth rates during this period increased exponentially, from 1 g week-1 at week 3 to 40 g week-1 at week 16. Thereafter, as the young left the pouch, marked changes were seen in carbohydrate and fat levels: by week 25, carbohydrate levels had fallen to 2 g 100 ml-1, and fat levels had risen to 26 g 100 ml-1. Protein levels increased moderately, reaching 15 g 100 ml-1 by week 25. Growth rates further increased during this period, to reach 60 g week-1 by week 25. Thus, trends in milk composition previously observed in Macropus species were observed also in the potoroo, suggesting a consistent pattern across the macropodid family. Carbohydrate levels in potoroo milk tend to be higher than in other macropodids, but total milk intake is as important as composition in determining growth rates.     

Establishment of nucleolar deficient sublines of PtK2 (Potorous tridactylis) by ultraviolet laser microirradiation

One of the two nucleoli of tetraploid PtK2 WA cells in early prophase was irradiated with an ultraviolet (UV) laser microbeam. The daughter cells that maintained the nucleolar deficiency were isolated and cloned. Five nucleolar deficient sublines of PtK2 WA were established, thus providing an experimental system to study the ribosomal gene-nucleolar organizer complex.     

Chromosomal banding patterns and karyotype evolution in three pig kidney cell strains (PK15, F and RP)

Some of the techniques used to obtain banding patterns in human karyotype are adapted here to three pig kidney cell strains (PK15, F and RP). These strains were established respectively in 1955, 1962 and 1969. The banding techniques used are: controlled heating, ASG technique, alkaline treatment and proteolytic digestion with trypsin or pronase. Knowing the specific banding of the pig karyotype, it has been possible to study the chromosomal rearrangements observed in the heteroploid cell strains. If the strain is old, the rearrangements are more numerous. However, they are the same as the ones usually described: in the three strains, one of the two chromosomes of each pair is retained unchanged as judged by its banding. The other chromosome is either present, lost or modified. It may constitute part of a marker chromosome.     

Molecular phylogenetic relationships of two extinct potoroid marsupials, Potorous platyops and Caloprymnus campestris (Potoroinae: Marsupialia)

Complete 12S rRNA and partial cytochrome b (cytb) gene sequences have been obtained from museum samples of two recently extinct potoroids-Potorous platyops and Caloprymnus campestris. Phylogenetic analyses based on these mitochondrial DNA sequences suggest that the broad-faced potoroo (P. platyops) was a close relative of the recently discovered Potorous longipes and the recently re-discovered Potorous gilberti. Although the extinct desert rat-kangaroo (C. campestris) was clearly resolved as a member of the subfamily Potoroinae, its precise relationships vis a vis other living potoroines are unclear. We confirmed that the rufous rat-kangaroo (Aepyprymnus rufescens) is sister to all living Bettongia species, but the molecular data provide no support for a sister relationship between A. rufescens and C. campestris as suggested by on the basis of four shared morphological characters. Molecular dating analyses suggest that the initial radiation of potoroinae seems to have occurred soon after its origin in the early Miocene. Within Potoroinae, C. campestris diverged from other taxa approximately 16 million years ago. P. platyops diverged from P. longipes+P. gilberti approximately 14-15 million years ago.     

Nucleoli and ploidy in Potorous cells (PTK2) in vitro

Most cells of the male PTK₂ cell line contain one nucleolus, and they are diploid. However, a proportion of cells have more than one nucleolus. Cells with two and three nucleoli were isolated and cloned into viable populations. Greater than 90% of the cells in these clonal populations maintained the abnormal nucleolar number of the originally isolated cell. Karyotype analysis of cells with two and three nucleoli demonstrated that the cells were respectively tetraploid and hexaploid. It was concluded that in PTK₂ cells, nucleolarity is a good index of ploidy even if the ploidy level is abnormal. Furthermore, long term monitoring of the tetraploid cells demonstrated virtually no tendency towards reversion to the diploid condition as suggested by other studies in Potorous.