Xkid, a Chromokinesin Required for Chromosome Alignment on the Metaphase Plate

In metaphase preparations from leucocytes of the toad, Bufo marinus, conspicuous secondary constrictions are present in the number 7 pair of chromosomes. These constrictions were considered to be the nucleolar organizers since they were associated with nucleoli during prophase. In 35 out of 60 individuals taken from natural populations, the homologous nucleolar organizers produced two equal-sized nucleoli and secondary constrictions (Group I animals). Pour animals (Group II) had only one very large secondary constriction in the majority of their metaphase preparations and an abnormally high frequency of cells containing one nucleolus. The remaining 21 animals (Group III) had unequal-sized constrictions in most of their metaphases but were more variable than the individuals of Groups I or II since they also had metaphases with two equal constrictions or only one constriction. The DNA from individuals of each group was hybridized with radioactive ribosomal RNA in order to correlate the size of nucleoli and constrictions with the amount of DNA (rDNA) homologous to ribosomal RNA. The two animals of Group II which were studied contained 0.056% of their genome homologous to ribosomal RNA a value considerably higher than those found for any of the animals of Groups I or III. These high values for rDNA coupled with the morphological appearance of the nucleolar homologues suggested a duplication of the nucleolar organizer in the homologue with the long constriction. The amount of rDNA in animals of Group I and III varied between 0.025 and 0.048% of the genome. Although the animals with unequal-sized constrictions (Group III) had generally lower contents of rDNA than those with equal-sized constrictions (Group I), the values overlapped between the two groups. Further evidence which correlates the size of nucleoli with the number of ribosomal RNA genes comes from studies with a “small nucleolar” mutant of the Mexican axolotl (Ambystoma mexicanum). Animals homozygous for this deletion were found to contain only 55% of the complement of rDNA present in the wild type. It is concluded that partial deletions and duplications of the nucleolar organizer as well as highly variable contents of rDNA are common in the genome of these amphibians.

Variation in the activity of nucleolar organizers and their ribosomal gene content.

The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.

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Role of Xklp3, a Subunit of the Xenopus Kinesin II Heterotrimeric Complex, in Membrane Transport between the Endoplasmic Reticulum and the Golgi Apparatus

That the ability of the nucleolar organizer to form nucleoli is under genetic control has also been demonstratedin hybrids of the plant genus Grepis. Each species of Crepis has its distinctive nucleolar organizer, but when brought into a common nucleus through hybridization, the organizers exhibit differential synthetic ability in the hybrid environment; only one of them will form a nucleolus, the other remaining inactive. When inactive, no nucleolus forms and the secondary constriction will be missing from the metaphase chromosomes. A number of Crepis species, therefore, can be graded on the competitive strength of their nucleolar organizers. An inactive organizer, on the other hand, regains its synthetic ability when placed back in a more favourable nuclear situation, and the metaphase chromosome will again show a secondary constriction.

/pdf/differential-amphiplasty-and-the-control-of-ribosomal-rna-niqrpu6720.pdf

Differential amphiplasty and the control of ribosomal RNA synthesis

The oytogenetic analysis of the genus Xenopus shows that X. laevis laevis, X. laevis petersi, X. laevis victorianus, X. (laevis) borealis, X. gilli, X. muelleri, and X. fraseri have chromosome numbers 2n=36; X. tropicalis has 20 (2n), X. (laevis) bunyoniensis 72 and X. ruwenzoriensis 108. This heterogeneity of the chromosome numbers is interesting as it represents new examples of polyploidy among Anurans. There are no big morphological differences among the karyotypes of the divers species, only the chromosomes with secondary constrictions vary considerably.

Chromosome complements of the genus Xenopus.

Secondary chromosomal constrictions are thought to be the loci of the genome which code for ribosomal RNA synthesis. Their metaphase length could depend on nucleolar size or level of functional activity in interphase or on gene content. Wild-type frogs and a frog heterozygous for the Oxford nucleolar mutation were studied to determine which possibility is more probable. The mutant was studied because its single nucleolus is larger than wild-type nucleoli, it has only one constriction, half as many ribosomal genes, but produces the same amount of ribosomal RNA. The results indicate (1) that constriction length depends on the amount of genome (whereas others have shown nucleolar size to be related to level of activity) and (2) that the deletion is limited to the constricted segment, supporting the view that the constriction is the nucleolar organizer. Also, metaphase constrictions are longer than expected from their DNA content.

R.W. Sherwin

Papers

The length of secondary chromosomal constrictions in normal individuals and in a nucleolar mutant of Xenopus laevis.