The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the ...apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). The dum-1 mutant was used as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with Cchlamydomonas reinhardtii mitochondrial DNA and a single colony was isolated from plates bombarded with Chlamydomonas smithii mitochondrial DNA. No colonies were seen on control plates. All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic Hpa1 and Xba1 restriction sites in the gene encoding subunit 1 of cytochrome oxidase fragments of the dum-1 recipient were not detected in any of the transformants.
To determine the association between type, chronicity, and severity of childhood hardships and smoking status during pregnancy, preterm birth (PTB), and low birth weight (LBW).
Prospective cohort ...study.
The National Child Development Study, a nationally representative study of births in Great Britain in 1958.
Four thousand eight-hundred sixty-five women with at least 1 singleton live birth.
Hardship during childhood, indicated by several variables, including financial/structural hardship, lack of parental interest in education, family dysfunction, violence/mental health issues, and family structure.
Smoking in pregnancy, LBW, and PTB.
A consistent and graded association was seen between all types of childhood hardships and smoking status during pregnancy (odds ratio OR for 4 or more hardships, 2.02; 95% confidence interval CI, 1.58-2.58; P < .001 for all comparisons). Most hardships were also associated with risk of LBW and PTB, with associations between number of hardships and both outcomes persisting after controlling for smoking status and adult social class (for LBW, OR, 1.51; 95% CI, 1.10-2.06; for PTB, OR, 1.44; 95% CI, 1.08-1.92).
Childhood hardships have an enduring impact on future pregnancy outcomes, in part through their association with smoking during pregnancy and adult socioeconomic position.
In this paper, we examine the effects of mutations in the 5'UTR of the chloroplast rps7 transcript of Chlamydomonas reinhardtii that reduce the stability of the mRNA. Five point mutants in the rps7 ...5'UTR were selected on the basis of their failure to accumulate reporter mRNA in Escherichia coli. Each of these mutations produces alterations in the predicted higher-order structures of the rps7 5'UTR that destabilize the mRNA. Cis-acting suppressors of these mutations have been selected in E. coli and in the C. reinhardtii chloroplast that restore message stability and function. No differences in RNA melting and reannealing profiles have been observed between wild type, original mutant, and suppressor 5'UTRs transcribed in vitro. Proteins of 32 kDa and 47 kDa that bind to the wild-type rps7 5'UTR are not detected by UV cross-linking assays performed with any of the mutant rps7 5'UTRs. However, binding of the 32-kDa protein is restored in the six suppressor mutants examined. This suggests that the 32-kDa protein may be involved in protecting the rps7 5'UTR and the attached coding region from digestion by ribonucleases. Alternatively, the binding site for the 32-kDa protein may be independently lost in the rearranged tertiary structure of the mutant 5'UTR that exposes the RNA to degradation and is restored in the suppressor mutants.
Expression of three chloroplast genes encoding proteins of different chloroplast complexes and the rRNA gene has been examined in cells having reduced numbers of chloroplast genomes as a result of ...growth in the presence of the thymidine analog 5-fluorodeoxyuridine. While accumulation of total mRNA for rp12 (ribosomal protein L-1), rbcL (ribulose bisphosphate carboxylase large subunit) and atpA (alpha-subunit of ATP synthase) declined with gene copy number, the levels of translatable mRNA and rates of synthesis of these three proteins were larggely unaffected. Accumulation of rRNA declined less precipitously than mRNA levels for the three proteins in response to the reduction in chloroplast genome number. Chlamydomonas appears to compensate for reductions in the number of chloroplast genomes at several different levels. Populations of cells with only one-fourth the wild-type amount of chloroplast DNA per cell on average have half the normal level of chloroplast ribosomes and nearly normal rates of CO2 fixation and levels of specific chloroplast encoded proteins. These results suggest that normal cells accumulate a large excess of transcripts for chloroplast genes and that levels of expression of these genes are regulated by posttranscriptional mechanisms
In Chlamydomonas reinhardtii, chloroplast genomes are normally transmitted by the mating type plus (mt+) parent and mitochondrial genomes by the mating type minus (mt-) parent. In this paper we ...describe three new nuclear mutations, designated mat-3-1 to -3, which are tightly linked to the mt+ allele and permit high transmission of chloroplast genomes from the mt- parent, but have no effect on transmission of mitochondrial genomes. We also show that mat-1, reported by others to be a nuclear mutation linked to mt- which promotes transmission of chloroplast genomes by the mt- parent, is probably a vegetative diploid since it contains both mt+ and mt- alleles. Vegetative diploids behave as if they are mt- with respect to mating, but possess a level of chloroplast gene transmission intermediate between that of haploid mt- and mt+ stocks.
A collection of very high-light-resistant,
VHL
R
, mutants that grow in 5% CO
2 under 1500 to 2000 μmol photons m
−2 s
−1 has been isolated from both the CC-125 wild-type strain of
Chlamydomonas ...reinhardtii and from the chloroplast
psbA mutant
A251L
* with an Ala to Leu substitution in the D1 protein. Neither of the parental strains survives long-term exposure to very high light intensities (VHL; above 1500 μmol photons m
−2 s
−1). This paper characterizes the genetic, physiological and biochemical properties of two nuclear mutants isolated from wild-type (
S4 and
S9), and two derived from
A251L
* (
L5 and
L30). All four mutants grow photoautotrophically in 5% CO
2 at rates similar to their respective parental strains under high light (HL; 600 μmol photons m
−2 s
−1) and maintain high growth rates under VHL. Total chlorophyll per biomass, or per cell, declines in both mutant and parental strains grown in HL compared with LL, and decreases further in mutants grown under VHL. All four
VHL
R
mutants have lower light and CO
2-saturated rates of photosynthetic O
2 evolution per total chlorophyll than wild-type or
A251L
* under low light (LL; 70 μmol photons m
−2 s
−1) or HL. The
VHL
R
mutants
S4 and
S9 show greater nonphotochemical fluorescence quenching than wild-type when grown and measured under HL or VHL. In contrast,
A251L
*,
L5 and
L30 exhibit low nonphotochemical quenching under all conditions. Although xanthophyll pigments, including lutein, are elevated on a chlorophyll basis in both wild-type and
S9 cells grown in HL compared to LL, they are unchanged on a biomass basis. The de-epoxidation state is very low even in HL. Preliminary analyses suggest that other
VHL
R
mutants behave similarly. Evidently, enhanced photoprotection via the xanthophyll cycle is not involved in survival of several different
VHL
R
mutants under saturating light and CO
2. Photosystem II properties and electron transfer rates in all
VHL
R
mutants grown in 5% CO
2 under LL do not differ from those of their respective parental strains. These experiments also demonstrate that slow photosystem II electron transfer in strain
A251L
* due to altered D1 structure, which is present in
L5 and
L30 isolated from this strain, does not preclude selection of
VHL
R
mutants. The ability of these mutants to survive under VHL in 5% CO
2 evidently involves processes other than photoprotection via the xanthophyll cycle and resistance to photodamage via alterations in D1 function.
The chloroplast (cp)-encoded CF1 ATPase beta-subunit gene (atpB) of Chlamydomonas reinhardtii and its flanking regions have been sequenced. The derived amino acid (aa) sequence is highly homologous ...to that of the beta-subunit gene in Escherichia coli, bovine heart mitochondria, and higher plant cp. In contrast to all other cp genomes, the CF1 epsilon subunit gene (atpE) does not lie at the 3' end of the atpB gene but maps to a position 92 kb away in the other single-copy region. Northern blots confirm that the beta subunit is not encoded as part of a dicistronic message as it is in higher plants. The region just upstream from the atpB gene in C. reinhardtii contains two small open reading frames (ORFs) and not the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase as is found in cp genomes of higher plants. No transcripts for either ORF were detected, but the codon usage in these ORFs as well as in the atpB gene follows the unique pattern of codon usage previously seen in other cp genes in C. reinhardtii.
PDF
