General practice as a career: insights for workforce policy.

Australia's general practitioners are working fewer hours, and many are leaving medical practice. Little is known about when and why experienced mid- and late-career GPs move away from clinical practice. Although career downsizing is often seen as an abrogation of vocation, it may reflect a desire to broaden work experiences within a constrained set of options.

Mitochondrial biogenesis in early mouse embryos: expression of the mRNAs for subunits IV, Vb, and VIIc of cytochrome c oxidase and subunit 9 (P1) of H(+)-ATP synthase.

The mouse egg contains about 90,000 mitochondria which undergo a buildup of mitochondrial cristae and increase in respiratory activity during cleavage. The mitochondrial DNA does not replicate during preimplantation development but is transcribed actively from the two-cell stage onward (Pikó and Taylor, 1987: Dev Biol 123:364-374). To gain further insight into mitochondrial biogenesis, we have now determined the steady state amounts of the mRNAs for the cytochrome c oxidase (COX) subunits IV, Vb and VIIc and the H(+)-ATPase subunit 9 (P1) (all encoded by nuclear genes) in slot hybridization experiments of total RNA from oocytes and early embryos.

Sequence of a mouse embryo cDNA clone encoding proteolipid subunit 9 (P1) of the mitochondrial H(+)-ATP synthase.

A cDNA clone to an abundantly expressed mRNA in cleavage stage mouse embryos has been sequenced and identified as encoding subunit 9 (P1) of the mitochondrial H(+)-ATP synthase. The deduced amino acid sequence of the mature subunit 9 protein differs in a single residue from the corresponding rat, ovine, bovine and human subunits.

Expression of ribosomal protein genes in mouse oocytes and early embryos.

The quantitative changes in the mRNAs for ribosomal proteins L7a, L18a, and S15 were assayed in slot hybridization experiments using labeled cRNA probes with total RNA from late growth-phase oocytes, ovulated eggs, and early embryos through the blastocyst stage. All three mRNAs showed a similar developmental pattern of prevalence, but their copy numbers per oocyte or embryo fluctuated according to developmental stage. There are on an average about 17,000 copies of each mRNA in the late growth-phase oocyte; this number drops to one-fifth to one-tenth in the ovulated egg and two-cell embryo but increases rapidly during cleavage to bout 25,000 in the eight-cell embryo and about 42,000 in the blastocyst.

Expression of the rig gene in mouse oocytes and early embryos.

A clone selected from a two-cell mouse embryo cDNA library has been sequenced and identified as rig cDNA. The rig gene codes for a highly conserved nuclear protein, which may have a general role in cell growth or replication (Shiga et al.: Proc Natl Acad Sci USA 87:3594, 1990).

Quantitative changes in cytoskeletal beta- and gamma-actin mRNAs and apparent absence of sarcomeric actin gene transcripts in early mouse embryos.

Actin is known to be synthesized both during oogenesis and in cleavage-stage embryos in mice. Cytoskeletal beta-actin appears to be the major component, followed by gamma-actin, but the synthesis of alpha-actin has also been inferred from protein electrophoretic patterns. We have studied the expression of cytoskeletal (beta- and gamma-) and sarcomeric (alpha-cardiac and alpha-skeletal) actin genes at the level of the individual mRNAs in blot hybridization experiments using isoform-specific RNA probes.

Studies of sequence heterogeneity of mitochondrial DNA from rat and mouse tissues: evidence for an increased frequency of deletions/additions with aging.

To obtain information on the extent of random nucleotide changes in mitochondrial DNA (mtDNA) from different organs of young adult and senescent Fischer 344 rats, the temperature of thermal denaturation (tm) was measured in (1) the native mtDNA cut at a single SstI site and (2) the reannealed duplexes formed after the initial melting of the mtDNA sample. No change was found between the two tm values in either young or senescent mtDNA, suggesting that the overall mismatch in nucleotide sequence in these samples was below the resolution of the method estimated at about 0.2%.

Patterns of mRNA prevalence and expression of B1 and B2 transcripts in early mouse embryos.

Considerable evidence indicates that the 2-cell stage is a critical period of mouse embryo development when a transition from maternal to zygotic genomic control takes place. The overall changes in the structure of the mRNA population as a result of this transition were explored using a random cDNA library of 69 clones derived from late 2-cell embryos. The prevalence of the cloned sequences was analysed by dot hybridization of the cDNA clones with labelled cDNA probes synthesized to poly(A)+ RNA from different stages of development from 1-cell through blastocyst.

Amounts of mitochondrial DNA and abundance of some mitochondrial gene transcripts in early mouse embryos.

The contents of mitochondrial DNA (mtDNA) and the steady-state amounts of 12 and 16 S mitochondrial rRNAs and the mRNAs for cytochrome c oxidase subunits I and II (COI and COII) were determined in dot hybridization experiments with cloned mtDNA fragments as probes during development from the one-cell to the blastocyst stage. The mtDNA content remained constant during this period at about 2.13 pg or 119,000 mtDNA molecules per embryo, suggesting an absence of mtDNA replication.

Structural and replicative forms of mitochondrial DNA in tissues from adult and senescent BALB/c mice and Fischer 344 rats.

Age-related changes in the structure and replication of mitochondrial DNA (mtDNA) were investigated in different organs from young adult (9-10 months' old) and senescent (28-29 months' old) BALB/c mice and Fischer 344 rats. Total mtDNA from brain, heart, kidney and liver was isolated by centrifugation in ethidium bromide-CsCl gradients and examined for the occurrence of complex forms and replicative intermediates by electron microscopy. The frequency of catenated mtDNA (interlinked molecules containing two or more circular units) varied from about 2.

Variation in the frequency of complex forms of mitochondrial DNA in different brain regions of senescent mice.

It was shown previously that the frequency of an aberrant form of mitochondrial DNA (mtDNA), double-sized circular molecules or circular dimers, increased significantly in the brain of senescent mice, to about 2% versus less than 0.1% in the brain of adult mice. To follow up these observations, we isolated total mtDNA from 6 different brain regions of 29-month-old male BALB/c mice and examined it for the occurrence of circular dimers and other complex forms by electron microscopy.

Amounts, synthesis, and some properties of intracisternal A particle-related RNA in early mouse embryos.

Early mouse embryos express two morphological subtypes of intracisternal A-type particles, one resembling those occurring in mouse tumors (referred to as IAP) and the other apparently specific for early embryos [referred to as IAP(epsilon)]. Using cloned fragments of IAP genes as labeled probes in dot-hybridization experiments, we detected IAP-related RNA sequences in mouse oocytes and preimplantation embryos. IAP RNA is relatively abundant in ovarian oocytes, is reduced in amount to approximately equal to 1/10th in the ovulated egg, and increases approximately equal to 100 times (from approximately equal to 1.

Quantitative aspects of RNA synthesis and polyadenylation in 1-cell and 2-cell mouse embryos.

Mouse embryos at the late 1-cell and late 2-cell stages were labelled with [3H]adenosine for periods of up to 320 min during which the specific activity of the ATP pool was constant. The time course of the molar accumulation of adenosine was calculated for tRNA, high-molecular-weight poly(A)- RNA and poly(A) tails versus internal regions of poly(A)+ RNA. Most of the adenosine incorporation into tRNA is due to turnover of the 3'-terminal AMP but some new synthesis of tRNA also appears to take place in both 1-cell and 2-cell embryos at a rate of about 0.

Poly(A) length, cytoplasmic adenylation and synthesis of poly(A)+ RNA in early mouse embryos.

The poly(A) content of early mouse embryos fluctuates widely: after a transient increase in the one-cell embryo, there is a 70% drop in the two-cell and an approximately fivefold increase between the two-cell and early blastocyst stages (L. Pikó and K. B.

Structural and replicative forms of mitochondrial DNA from human leukocytes in relation to age.

The structure and replication of human leukocyte mitochondrial DNA (mtDNA) was investigated in healthy young adult males (23--37 years old), middle-aged males (42--52 years old) with secondary polycythemia, and elderly males (80--89 years old) who exhibited different degrees of age-related disease syndromes. The distribution of the various cell types within the white cell population was within normal limits in all samples. Total mtDNA was isolated in ethidium bromide--CsCl gradients and examined by electron microscopy after spreading by the aqueous and formamide techniques.

Complex forms and replicative intermediates of mitochondrial DNA in tissues from adult and senescent mice.

The occurrence and types of complex forms and replicative intermediates of mitochondrial DNA (mtDNA) were investigated in tissues from C57BL/6J mice aged 10-11 months or 29-30 months. Total mtDNA from brain, heart, kidney and liver was isolated in ethidium bromide-CsCl gradients and examined by electron microscopy after aqueous or formamide spreading. Contour length measurements indicated no difference in the monomer size of mtDNA according to either tissue or donor age.

Complex mitochondrial DNA in animal thyroids. A comparative study.

1. The frequency of circular dimers and catenanes was determined in thyroid mitochondrial DNA (mtDNA) from rabbits, mice, pigs, sheep and cattle. 2.