Age bias in survey sampling and implications for estimating HIV prevalence in men who have sex with men: insights from mathematical modelling.

Respondent-driven sampling (RDS) is widely used to estimate HIV prevalence in men who have sex with men (MSM). Mathematical models that are calibrated to these data may be compromised if they fail to account for selection biases in RDS surveys. To quantify the potential extent of this bias, an agent-based model of HIV in South Africa was calibrated to HIV prevalence and sexual behaviour data from South African studies of MSM, first reweighting the modelled MSM population to match the younger age profile of the RDS surveys (age-adjusted analysis) and then without reweighting (unadjusted analysis). The model estimated a median HIV prevalence in South African MSM in 2015 of 34.6% (inter-quartile range (IQR): 31.4-37.2%) in the age-adjusted analysis, compared with 26.1% (IQR: 24.1-28.4%) in the unadjusted analysis. The median lifetime risk of acquiring HIV in exclusively homosexual men was 88% (IQR: 82-92%) in the age-adjusted analysis, compared with 76% (IQR: 64-85%) in the unadjusted analysis. These results suggest that RDS studies may under-estimate the exceptionally high HIV prevalence rates in South African MSM because of over-sampling of younger MSM. Mathematical models that are calibrated to these data need to control for likely over-sampling of younger MSM.

G1 events and the regulation of genes for S-phase enzymes.

The genes for S-phase enzymes are expressed at low levels in quiescent mammalian cells but at high levels during DNA replication. Regulation occurs at multiple levels by mechanisms that are different for each gene. Current research is focused on identifying the control elements and trans-acting factors for each gene and establishing relationships between these regulatory mechanisms and the G1 signal transduction pathway.

The role of immunity in the epidemiology of gonorrhoea, chlamydial infection and trichomoniasis: insights from a mathematical model.

Most mathematical models of sexually transmitted infections (STIs) assume that infected individuals become susceptible to re-infection immediately after recovery. This paper assesses whether extending the standard model to allow for temporary immunity after recovery improves the correspondence between observed and modelled levels of STI prevalence in South Africa, for gonorrhoea, chlamydial infection and trichomoniasis. Five different models of immunity and symptom resolution were defined, and each model fitted to South African STI prevalence data. The models were compared in terms of Bayes factors, which show that in the case of gonorrhoea and chlamydial infection, models that allow for immunity provide a significantly better fit to STI prevalence data than models that do not allow for immunity. For all three STIs, estimates of the impact of changes in STI treatment and sexual behaviour are significantly lower in models that allow for immunity. Mathematical models that do not allow for immunity could therefore overestimate the effectiveness of STI interventions.

The next generation: Pregnancy in adolescents and women living with perinatally acquired HIV in South Africa.

BACKGROUND: An increasing number of girls living with perinatally acquired HIV (PHIV) are reaching adolescence and adulthood and becoming pregnant. Youth living with PHIV (YLPHIV) may have HIV-associated infections/complications, long-term exposure to antiretroviral treatment (ART), drug resistance and increased psychosocial challenges, which may adversely affect pregnancy outcomes. There is a lack of published studies on pregnancy in YLPHIV in sub-Saharan Africa. Objectives. To describe characteristics of pregnant South African (SA) YLPHIV and their pregnancy outcomes. METHODS: We retrospectively identified pregnancies in YLPHIV, who were diagnosed with HIV when they were <12 years old and before their first pregnancy (as a proxy for perinatal route of infection), from routinely collected data in Western Cape Province, SA (2007 - 2018). We combined these with pregnancies from a Johannesburg cohort of YLPHIV. Results. We identified 258 pregnancies among 232 females living with likely PHIV; 38.8% of pregnancies occurred in YLPHIV ≤16 years old, 39.1% at age 17 - 19 years and 22.1% at age ≥20 years. In recent years, a steady increase in the number of pregnancies in YLPHIV was noted; more than two-thirds occurred during 2016 - 2018. ART was commenced prior to pregnancy in 84.9% of YLPHIV, during pregnancy in 6.6% and was not commenced by pregnancy end date in 8.5%. Of the pregnancies in young women with documented outcomes (88.8%; n=229), 80.3% were live births, 14.4% terminations, 3.1% miscarriages and 2.2% stillbirths. Mother-to-child transmission of HIV occurred in 2.2% of infants, 75.3% were uninfected when last tested and 22.6% had unknown HIV status. Among YLPHIV with CD4 counts available within 12 months of pregnancy end date (n=202), 20.3% had a CD4 count <200 cells/µL, 43.1% CD4 count 200 - 499 cells/µL and 36.6% CD4 count ≥500 cells/µL. Among those with a viral load (VL) available within 12 months of pregnancy end date (n=219), 66.7% had a VL <400 copies/mL, 5.0% VL 400 - 999 copies/mL and 28.3% VL ≥1 000 copies/mL. Of 186 neonates, 20.4% were preterm deliveries (<37 weeks' gestation). Among neonates with known birthweight (n=176), the mean birthweight was 2 900 g (95% confidence interval (CI) 2 747 - 2 935 g) and 20.5% had a low birthweight (<2 500 g). One congenital malformation (musculoskeletal) and 2 neonatal deaths were recorded. CONCLUSIONS: In recent years, the number of pregnancies in YLPHIV has increased. A considerable proportion of pregnancies occurred in YLPHIV ≤16 years old. A high proportion of pregnancies was electively terminated. The prevalence of elevated VL and poor immunological status among pregnant YLPHIV is concerning.

Uptake and acceptability of medical male circumcision among young males in two culturally distinct settings in South Africa: A longitudinal, community-based study (the MACHO study).

BACKGROUND: Young South Africans experience high rates of HIV infection. While nationally scaled medical male circumcision (MMC) can help to curb HIV infection rates in countries such as South Africa (SA), MMC uptake has not been consistent or universal, suggesting variable acceptability among men. Both MMC and traditional male circumcision (TMC) are practised in SA. For male circumcision to be most effective for HIV prevention, it should be performed prior to sexual debut with complete removal of the foreskin. OBJECTIVES: The MACHO (Male Adolescent Choices for HIV Prevention Options) study investigated uptake of and preference for MMC v. TMC in two culturally distinct settings in SA. METHODS: This observational, longitudinal, cohort study investigated circumcision preferences and uptake in 100 males (aged 14 - 17 years) and their legal guardians in Cape Town (Western Cape Province) and Soweto (Gauteng Province). Data were collected via surveys administered every 4 months over a 24-month period. RESULTS: A total of 100 uncircumcised adolescent boys (Cape Town n=50, Soweto n=50; mean (interquartile range) age 15 (14 - 16) years) and their guardians were enrolled. At baseline, 42 boys from Soweto (84%) and none from Cape Town expressed a preference for MMC over TMC. Sowetan participants were more likely to elect circumcision (MMC n=11 (22%), TMC n=1 (2%)) than those from Cape Town (TMC n=1 (2%), MMC n=0) over 13.6 months of follow-up (hazard ratio 18.9; 95% confidence interval 2.37 - 150.71; p=0.006). CONCLUSIONS: MMC was the preferred option for young men in Soweto compared with those in Cape Town, and this translated into practice. Despite knowledge of the benefits of early MMC, many participants delayed uptake, potentially reducing the MMC benefits before sexual debut. Programmes promoting circumcision should consider the influence of local practices. To realise full HIV prevention benefits, efforts should be made to ensure that circumcision is promoted, and that all circumcision is safe, performed prior to sexual debut, and contextually responsive.

High incidence of tuberculosis among HIV-infected infants: evidence from a South African population-based study highlights the need for improved tuberculosis control strategies.

BACKGROUND: There are limited population-based estimates of tuberculosis incidence among human immunodeficiency virus (HIV)-infected and HIV-uninfected infants aged < or =12 months. We aimed to estimate the population-based incidence of culture-confirmed tuberculosis among HIV-infected and HIV-uninfected infants in the Western Cape Province, South Africa. METHODS: The incidences of pulmonary, extrapulmonary, and disseminated tuberculosis were estimated over a 3-year period (2004-2006) with use of prospective representative hospital surveillance data of the annual number of culture-confirmed tuberculosis cases among infants. The total number of HIV-infected and HIV-uninfected infants was calculated using population-based estimates of the total number of live infants and the annual maternal HIV prevalence and vertical HIV transmission rates. RESULTS: There were 245 infants with culture-confirmed tuberculosis. The overall incidences of tuberculosis were 1596 cases per 100,000 population among HIV-infected infants (95% confidence interval [CI], 1151-2132 cases per 100,000 population) and 65.9 cases per 100,000 population among HIV-uninfected infants (95% CI, 56-75 cases per 100,000 population). The relative risk of culture-confirmed tuberculosis among HIV-infected infants was 24.2 (95% CI, 17-34). The incidences of disseminated tuberculosis were 240.9 cases per 100,000 population (95% CI, 89-433 cases per 100,000 population) among HIV-infected infants and 14.1 cases per 100,000 population (95% CI, 10-18 cases per 100,000 population) among HIV-uninfected infants (relative risk, 17.1; 95% CI, 6-34). CONCLUSIONS: This study indicates the magnitude of the tuberculosis disease burden among HIV-infected infants and provides population-based comparative incidence rates of tuberculosis among HIV-infected infants. This high risk of tuberculosis among HIV-infected infants is of great concern and may be attributable to an increased risk of tuberculosis exposure, increased immune-mediated tuberculosis susceptibility, and/or possible limited protective effect of bacille Calmette-Guérin vaccination. Improved tuberculosis control strategies, including maternal tuberculosis screening, contact tracing of tuberculosis-exposed infants coupled with preventive chemotherapy, and effective vaccine strategies, are needed for infants in settings where HIV infection and tuberculosis are highly endemic.

Disseminated bacille Calmette-Guérin disease in HIV-infected South African infants.

OBJECTIVE: To determine the population-based incidence of disseminated bacille Calmette-Guérin (BCG) disease in HIV-infected infants (aged

Changes in RNA in relation to growth of the fibroblast. IV. Alterations in theproduction and processing of mRNA and rRNA in resting and growing cells.

In previous reports, it was shown that both the concentration and rate of production of rRNA and mRNA were greater in growing than in resting 3T6 fibroblasts. Studies on isolated nuclei indicated that ribosomal RNA production is apparently controlled at the level of transcription. In contrast, hnRNA, the putative precursor of mRNA, appeared to be synthesized at the same rate in resting and growing cells. This finding was unexpected and has been tested in several ways. In this report, we show by an independent method that the relative rate of production of mRNA compared to hnRNA is several-fold higher in growing than in resting cells. However, the kinetics of processing of mRNA appear unchanged. This result suggests either that mRNA arises from a small subfraction of hnRNA or that the efficiency of processing of the hnRNA precursor is an important control mechanism which determines mRNA production ingrowing and resting states. Comparison of the initial rates of labeling of hnRNA and cytoplasmic message gives the efficiency with which the cytoplasmic mRNA is produced from nucleoplasmic RNA. The very low efficiency (3-4% in growing and 1-2% in resting cells) suggests that not every hnRNA molecule gives rise to a cytoplasmic message. In contrast to the similar kinetics of mRNA production in resting and growing states, processing of ribosomal RNA is much slower in the resting state and the emergence time for 28S RNA from nucleolus is greatly lengthened.

Nuclear magnetic resonance measurement of oil "unsaturation" in single viable corn kernels.

High-resolution nuclear magnetic resonance spectroscopy has been used to demonstrate the feasibility of determining iodine value and average molecular weight of oil in individual corn kernels. The procedure is rapid and nondestructive. Depending on heritability of individual fatty acids, this technique may greatly increase selection efficiency in breeding programs to alter the fatty acid composition of corn oil.

Splicing signals are required for S-phase regulation of the mouse thymidylate synthase gene.

The thymidylate synthase (TS) gene is expressed at a much higher level in cells undergoing DNA replication than in nondividing cells. In growth-stimulated mammalian cells, TS mRNA content increases 10 to 20-fold as cells progress from G1 through S phase. However, the rate of transcription of the TS gene does not increase during this interval, indicating that the gene is regulated at the posttranscriptional level. We have shown that both the promoter of the mouse TS gene and TS introns are necessary (although neither is sufficient) for S-phase-specific regulation of TS mRNA content. In the present study, we examined in more detail the role of introns in regulating TS mRNA levels in growth-stimulated cells. TS minigenes that contain normal or modified introns were stably transfected into mouse 3T6 fibroblasts, and the regulation of the minigenes was compared with that of the endogenous TS gene. TS minigenes that contain TS intron 1 or 2 maintain S-phase regulation. Deletion of most of the interior of the introns had only minor effects on regulation. However, when splicing of the intron was inhibited by alteration of the splice donor and acceptor sites, the minigene was expressed at a constant level following growth stimulation. Minigenes consisting of the TS promoter linked to either a luciferase or a human beta-globin indicator gene were growth regulated when spliceable introns were included in the minigenes. However, when the introns were eliminated, the minigenes were expressed at a constant level. These observations indicate that the splicing reaction itself, rather than a control sequence within the intron, is important for growth-regulated expression of the TS gene. Possible mechanisms to account for the dual requirement for the TS promoter and intron splicing for proper regulation of the TS gene are discussed.

Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter.

The mouse thymidylate synthase promoter lacks a TATA box and initiates transcription at many sites across a 90-nucleotide initiation window. We showed previously that wild-type promoter activity is maintained with a promoter that extends only 13 nucleotides upstream of the first start site. G/A-rich and G/C-rich promoter elements were identified in the vicinity of the first transcriptional start site. The goals of the present study were to determine whether there are additional promoter elements in the initiation window and to determine why transcription initiates across such a broad region. Minigenes containing a variety of substitution, deletion, and insertion mutations in the promoter region were transfected into cultured cells, and the effects on expression and the pattern of start sites were determined. The results indicate that there are no additional promoter elements downstream of the G/C box. The boundaries of the transcription window are established by elements near the 5' end of the window, whereas the pattern of start sites is determined by sequences within the window. The promoter lacks an initiator element. When an initiator element was inserted, transcription initiated predominantly at the position directed by the initiator when it was inserted within the initiation window but not when it was inserted immediately upstream of the window.

Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

The thymidylate synthase (TS) gene is expressed at much higher levels in proliferating cells than in quiescent cells. We have been studying the sequences that are important for regulating the mouse TS gene. We previously showed that DNA sequences upstream of the essential promoter elements as well as downstream of the ATG codon are both necessary (but neither is sufficient) for normal regulation in growth-stimulated cells. In the present study, we examined the possible roles of the coding region, polyadenylation signal, and introns as downstream regulatory elements. Minigenes consisting of 1 kb of the TS 5'-flanking region, the coding region (with or without various introns at their normal locations), and polyadenylation signals from the TS gene, the human beta-globin gene, and the bovine growth hormone gene were stably transfected into wild-type mouse 3T6 cells. Minigenes that contained introns 5 and 6, 1 and 2, or 1 alone were regulated regardless of which polyadenylation signal was included. A minigene that contained an internally deleted version of intron 1 was also regulated in response to growth stimulation. However, when all introns were omitted, there was little if any change in the level of minigene expression as cells progressed from G1 through S phase. These observations indicate that TS introns contain sequences that are necessary for normal growth-regulated expression of the mouse TS gene. These sequences appear to be associated with sequences that are important for splicing and to function in cooperation with upstream regulatory elements to bring about normal S-phase-specific expression.

The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells.

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.

Thymidylate synthetase overproduction in 5-fluorodeoxyuridine-resistant mouse fibroblasts.

We describe the isolation and characterization of a series of 5-fluorodeoxyuridine (FdUrd)-resistant mouse 3T6 cell lines that overproduce thymidylate synthetase (TS) by up to 50-fold compared with the parental cells. The resistant cells were selected by growing 3T6 cells or a methotrexate-resistant 3T6 cell line (M50L3, isolated previously in our laboratory) in gradually increasing concentrations of FdUrd. Uridine and cytidine were included in the culture medium to reduce toxicity from metabolic products of FdUrd. Cells that were resistant to the drug by virtue of loss of thymidine kinase activity were eliminated by selection in medium containing hypoxanthine, methotrexate, and thymidine. M50L3 cells were found to adapt to FdUrd more readily than 3T6 cells. A number of clones were isolated that were able to grow in the presence of 3 microM (M50L3 derived) or 0.3 microM (3T6 derived) FdUrd. Several were found to overproduce TS by 10 to 50-fold compared with normal 3T6 cells. All were found to have thymidine kinase activity, although the enzyme level was significantly reduced in some clones. The overproduced TS was inactivated by 5-fluorodeoxyuridylic acid at the same concentration as the enzyme from 3T6 cells. TS was purified from the LU3-7 clone (50-fold overproducer) by affinity chromatography on methotrexate-polyacrylamide. The monomer molecular weight was about 38,000, which was the same as the molecular weight of the monomer in 3T6 cells. The overproduction trait was gradually lost (half-life, 3 weeks) when LU3-7 cells were grown in the absence of FdUrd. The overproducing cells will provide an abundant supply of TS and (very likely) its mRNA and may serve as a convenient model system for detailed studies of the regulation of TS gene expression during the cell cycle.

The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites.

The promoter region of the mouse thymidylate synthase gene was analyzed by deletion and site-directed mutagenesis. Elimination of an upstream Sp1 element reduced expression threefold, whereas elimination of an adenovirus upstream stimulatory factor element had little effect. All of the upstream elements that are essential for promoter activity are located within 22 nucleotides of the first transcriptional initiation site.

Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse cell line.

We studied the content and metabolism of thymidylate synthase mRNA in cultured mouse fibroblasts that were undergoing a serum-induced transition from the resting to growing state. The studies were performed with a 5-fluorodeoxyuridine-resistant 3T6 cell line (LU3-7) that over produces the enzyme and its mRNA about 50-fold and that regulates the expression of the thymidylate synthase gene in the same manner as the parental cell line. We have previously shown that the rate of synthesis of thymidylate synthase increases at least ninefold when the serum-stimulated cells traverse the S phase. Here we show, by Northern blot analysis, that thymidylate synthase mRNA increased 20- to 40-fold as cells progressed from resting to late S phase. About 85% of poly(A)+ thymidylate synthase mRNA was associated with polysomes at all times. The increase in thymidylate synthase poly(A)+ mRNA content was the result of an eightfold increase in the rate of production of this species, as shown by pulse-labeling studies. Pulse-chase analysis revealed that the half-life of thymidylate synthase poly(A)+ mRNA was similar in resting (9 h) and growing (7 h) cells. The rate of transcription of the thymidylate synthase gene, as determined in isolated nuclei, increased only by a factor of three to four during the S phase. Since the content of the message increased to a much greater extent than the rate of transcription of the gene, posttranscriptional controls must also play a role in regulating the content of thymidylate synthase mRNA under these conditions. Our results suggest that the cell may regulate the distribution of thymidylate synthase mRNA between a relatively stable poly(A)+ RNA species and a labile poly(A)- RNA species.

Regulation of histone mRNA production and stability in serum-stimulated mouse 3T6 fibroblasts.

We measured the content and metabolism of histone mRNA in mouse 3T6 fibroblasts during a serum-induced transition from the resting to growing state. The content of several histone H3 and H2b mRNAs was measured by an S1 nuclease procedure. All of these increase in parallel by a factor of about 50 during S phase. However, the rate of H3 gene transcription increased only fivefold during this period, as determined in an in vitro transcription assay. This suggests that histone mRNA content is also controlled at the posttranscriptional level. When resting cells were serum stimulated in the presence of cytosine arabinoside, the rate of H3 gene transcription increased to about the same extent as that in control-stimulated cells. However, cytoplasmic H3 mRNA content increased only five to seven-fold. The half-life of H3 mRNA during S phase was about 4 to 5 h. When cytosine arabinoside was added to cells in the S phase, the half-life of the message decreased to about 15 min. The rapid turnover of H3 mRNA was prevented when the drug was added in the presence of cycloheximide or puromycin. The rate of H3 gene transcription decreased by only 35% after treatment with cytosine arabinoside. These results suggest that H3 gene transcription is not tightly coupled to DNA replication but is controlled temporally during the resting to growing transition. However, there is a correlation between the rate of DNA synthesis and the stability of histone H3 mRNA.

Regulation of ribosomal protein mRNA content and translation in growth-stimulated mouse fibroblasts.

When resting (G0) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates of synthesis of rRNA and ribosomal proteins increase, resulting in an increase in ribosome content beginning about 6 h after stimulation. In this study, we monitored the content, metabolism, and translation of ribosomal protein mRNA (rp mRNA) in resting, exponentially growing, and serum-stimulated 3T6 cells. Cloned cDNAs for seven rp mRNAs were used in DNA-excess filter hybridization studies to assay rp mRNA. We found that about 85% of rp mRNA is polyadenylated under all growth conditions. The rate of labeling of rp mRNA relative to total polyadenylated mRNA changed very little after stimulation. The half-life of rp mRNA was about 11 h in resting cells and about 8 h in exponentially growing cells, values which are similar to the half-lives of total mRNA in resting and growing cells (about 9 h). The content of rp mRNA relative to total mRNA was about the same in resting and growing 3T6 cells. Furthermore, the total amount of rp mRNA did not begin to increase until about 6 h after stimulation. Since an increase in rp mRNA content did not appear to be responsible for the increase in ribosomal protein synthesis, we determined the efficiency of translation of rp mRNA under different conditions. We found that about 85% of pulse-labeled rp mRNA was associated with polysomes in exponentially growing cells. In resting cells, however, only about half was associated with polysomes, and about 30% was found in the monosomal fraction. The distribution shifted to that found in growing cells within 3 h after serum stimulation. Similar results were obtained when cells were labeled for 10.5 h. About 70% of total polyadenylated mRNA was in the polysome fraction in all growth states regardless of labeling time, indicating that the shift in mRNA distribution was species specific. These results indicate that the content and metabolism of rp mRNA do not change significantly after growth stimulation. The rate of ribosomal protein synthesis appears to be controlled during the resting-growing transition by an alteration of the efficiency of translation of rp mRNA, possibly at the level of protein synthesis initiation.

Delayed processing of dihydrofolate reductase heterogeneous nuclear RNA in amino acid-starved mouse fibroblasts.

We have studied the metabolism of dihydrofolate reductase (DHFR) RNA in cells synchronized in the G1 phase of the cell cycle by starvation for isoleucine and glutamine. The relative content and stability of DHFR mRNA and the relative rate of transcription of the DHFR gene are similar in starved and exponentially growing cells. However, the relative rate of labeling of DHFR mRNA is about three times lower in starved cells than in exponentially growing cells. When the starved cells are stimulated to reenter the cell cycle by feeding them with complete medium, the relative rate of labeling of DHFR mRNA increases about fourfold within 6 h. However, the relative rate of transcription of the DHFR gene changes very little during this period. Continuous labeling experiments show that starved cells convert DHFR heterogeneous nuclear RNA into cytoplasmic DHFR mRNA much more slowly than serum-limited or exponentially growing cells. Pulse-chase experiments show that DHFR mRNA sequences contained in DHFR heterogeneous nuclear RNA appear to be conserved in starved cells. In addition, the content of DHFR RNA sequences in the nuclei of starved cells is about three times greater than that in exponentially growing cells. Delayed processing of DHFR heterogeneous nuclear RNA is also observed when exponentially growing cells are treated with inhibitors of protein synthesis. Our results suggest that, although delayed processing leads to a decrease in the initial labeling rate of DHFR mRNA, it does not result in a decrease in the actual rate of production of the message.

Stable inhibition of human thymidylate synthase expression following retroviral introduction of an siRNA gene.

Thymidylate synthase (TS) is an essential enzyme that synthesizes thymidylic acid in the de novo biosynthetic pathway. Inhibiting TS enzyme activity with substrate or cofactor analogs leads to inhibition of DNA replication and cell death. For this reason, TS is an important target enzyme for cancer chemotherapeutic drugs. We describe an alternative approach to reducing cellular TS enzyme activity using short interfering RNA (siRNA) technology to lower TS mRNA levels. Plasmids that direct the synthesis of siRNAs that target nucleotides 898-916 and 965-983 (relative to the A of the translational start codon) of human TS mRNA were highly effective at reducing TS enzyme levels in transient transfection assays. Infection of HeLa cells with retroviruses that contain the effective siRNA genes led to a stable 80-95% reduction of TS enzyme and mRNA. A similar percent reduction in TS expression was observed in a cell line that overproduces TS enzyme 100-fold due to TS gene amplification. Cells that exhibited the greatest reduction in TS enzyme level grew poorly in medium that lacked thymidine. These observations suggest that siRNA approaches may provide an alternative therapeutic strategy to reduce TS enzyme levels.