Karlee Babcock

Nuclear DNA content and chromosome numbers in the myxomycete Physarum polycephalum.

Abstract An improved method is described for making chromosome spreads of the plasmodium of the myxomycete, Physarum polycephalum . It consists of isolating metaphase nuclei, spreading the chromosomes with hot lactic acid, and staining with acetic-orcein. Most sublines derived from the Backus Wis 1 sclerotium had about 1 pg of DNA per nucleus, and had nuclei with 50 and 75 chromosomes in both the growing and sporulating plasmodium. Mature spores contained 0.6 pg of DNA, and hatching amoebae had 20–25 chromosomes and 0.6 pg of DNA. Plasmodia of the homothallic Colonia strain had a nuclear DNA content of about 1 pg, and had 35–40 chromosomes during growth and sporulation. Polyploid plasmodial sublines were found which had 1.5 and 3 times the normal DNA content and chromosome number. The polyploid sublines had the same plasmodial protein:DNA and RNA:DNA ratios as normal cultures. DNA content of nuclei varied directly with nuclear surface area. Ploidy was determined by the parent amoebae and therefore can serve as a genetic marker. A simple technique is given for completing the life cycle of P. polycephalum axenically. Germinating spores are plated without bacteria on one-tenth strength semidefined plasmodial growth medium, containing 2% agar. Plasmodia are visible in 2–4 days.

Changes in the DNA methylation profile of the rat H19 gene upstream region during development and transgenic hepatocarcinogenesis and its role in the imprinted transcriptional regulation of the H19 gene

Monoallelic expression of the imprinted H19 and insulin‐like growth factor‐2 (Igf2) genes depends on the hypomethylation of the maternal allele and hypermethylation of the paternal allele of the H19 upstream region. Previous studies from our laboratory on liver carcinogenesis in the F1 hybrid of Fischer 344 (F344) and Sprague–Dawley Alb SV40 T Ag transgenic rat (SD) strains revealed the biallelic expression of H19 in hepatomas. We undertook a comparative study of the DNA methylation status of the upstream region of H19 in fetal, adult, and neoplastic liver. Bisulfite DNA sequencing analysis of a 3.745‐kb DNA segment extending from 2950 to 6695 bp of the H19 upstream region revealed marked variations in the methylation patterns in fetal, adult, and neoplastic liver. In the fetal liver, equal proportions of hyper‐ and hypomethylated strands revealed the differentially methylated status of the parental alleles, but in neoplastic liver a pronounced change in the pattern of methylation was observed with a distinct change to hypomethylation in the short segments between 2984 and 3301 bp, 6033–6123 bp, and 6518–6548 bp. These results indicated that methylation of all cytosines in this region may contribute to the imprinting status of the rat H19 gene. This phenomenon of differential methylation‐related epigenetic alteration in the key cis‐regulatory domains of the H19 promoter influences switching to biallelic expression in hepatocellular carcinogenesis. Similar to mouse and human, we showed that the zinc‐finger CCTCC binding factor (CTCF) binds to the unmethylated CTCF binding site in the upstream region to influence monoallelic imprinted expression in fetal liver. CTCF does not appear to be rate limiting in fetal, normal, and neoplastic liver. 3′ to the CTCF binding sites, another DNA region exhibits methylation of CpGs in both DNA strands in adult liver, retention of the imprint in fetal liver, and complete demethylation in neoplastic liver. In this region is also a putative binding site for a basic helix‐loop‐helix leucine‐zipper transcription factor, TFEB. The differential CpG methylation seen in the adult that involves the TFEB binding site may explain the lack of expression of the H19 gene in adult normal liver. Furthermore, these findings demonstrate that the loss of imprinting of the H19 gene in hepatic neoplasms of the SD Alb SV40 T Ag transgenic rat is directly correlated with and probably the result of differential methylation of CpG dinucleotides in two distinct regions of the gene that are within 4 kb 5′ of the transcription start site. Cytogenetic analysis of hepatocytes in the transgenic animal prior to the appearance of nodules or neoplasms indicates a role of such loss of imprinting in the very early period of neoplastic development, possibly the transition from the stage of promotion to that of progression.

Biallelic expression of the H19 gene during spontaneous hepatocarcinogenesis in the albumin SV40 T antigen transgenic rat

Previous studies in this laboratory have demonstrated that the earliest cytogenetic alteration in the development of hepatic neoplasms in a transgenic strain of rats bearing the albumin Simian virus 40 T antigen (Alb SV40 T Ag) construct was a duplication of the chromosome 1q4.1‐1q4.2 band. In this region, in the rat genome a cluster of linked imprinted genes occurs. One of these imprinted genes, H19, which is expressed in fetal liver but not in adult liver, was found to be expressed in virtually all neoplasms investigated. A single‐nucleotide polymorphic marker in the H19 coding sequence was identified in two rat strains and utilized for the investigation of H19 imprinting. Our results reveal monoallelic expression of the maternal gene in fetal liver, but biallelic expression of the H19 gene in liver neoplasms, thus demonstrating the basis for the deregulation of the imprinted gene expression during hepatocarcinogenesis. These results suggest that the loss of genomic imprinting of the H19 gene found in the liver neoplasms of the Alb SV40 T Ag rat may result not from allelic loss, but from adverse changes in the epigenetic imprints present in the 5′‐upstream region of the H19 promoter of the parental alleles.

Alterations in specific gene expression and focal neoplastic growth during spontaneous hepatocarcinogenesis in albumin-SV40 T antigen transgenic rats.

Transgenic rats containing the mouse albumin promoter and enhancer directing the expression of simian virus (SV40) T antigen (T Ag) exhibited a 100% incidence of hepatic neoplasms by 24–36 wk of age. These transgenic rats exhibited expression of large T Ag and c‐myc protein within focal basophilic lesions and nodules, but not in surrounding hepatocytes. At 24 wk of age, female TG+ rats exhibited a significantly greater number of lesions and a much greater percentage of the liver occupied by TG+ focal hepatic lesions than did their male TG+ littermates. Previous studies on these animals [Sargent et al., Cancer Res 1997;57:3451–3456] demonstrate that at 12 wk of age approximately one‐third of metaphases in hepatocytes exhibit a duplication of the 1q3.7‐1q4.1 region of rat chromosome 1, with the smallest common region of duplication being that of 1q4.1. Duplication of the 1q3.7‐1q4.3 region is also noted in many primary hepatic neoplasms resulting from the multistage model of Initiation‐Promotion‐Progression (IPP) [Sargent et al., Cancer Res 1996;56:2985–2991]. This region is syntenic with human 11p15.5 and mouse 7ter, which have been implicated in the development of specific neoplasms. Within the syntenic region was a cluster of imprinted genes whose expression we investigated in livers and neoplasms of TG+ rats. H19 was expressed in almost all of the neoplasms, but not in normal adult liver cells. Igf2 expression was detected in the majority of hepatic neoplasms of female TG+ rats, but in a relatively smaller number of neoplasms of TG+ males. The expression of p57Kip2 (Kip2), a cyclin‐dependent kinase inhibitor that was also in the imprinted region, exhibited some variable increased expression predominantly in hepatic neoplasms from livers of female TG+ rats. Other imprinted genes within the imprinted gene cluster—insulin II (Ins2), Mash2 (which codes for a basic helix‐loop‐helix transcription factor), and Kvlqt1 (coding for a component of a potassium transport channel)—showed no consistently different expression from that seen in normal hepatocytes. Another gene, also located on the long arm of chromosome 1, that showed changes was the ribonucleotide reductase M1 subunit (Rrm1), in which an increase in its expression was found. This was seen in hepatic neoplasms of TG+ rats of both sexes compared with surrounding normal‐appearing liver. Because hepatic neoplasms developing in livers of rats treated with chemical carcinogens commonly exhibit an increased expression of c‐myc mRNA, expression of this gene was investigated in focal lesions and livers of TG+ rats, although c‐myc was not located on chromosome 1. c‐myc mRNA was increased in focal lesions, nodules, and neoplasms in both male and female TG+ rats compared with adult and surrounding liver. Immunostaining for c‐myc protein demonstrated detectable levels in isolated single cells as well as focal lesions and neoplasms. Thus, the enhanced c‐myc expression, common to all hepatic neoplasms in this system, coupled with enhanced expression of Igf2 in female TG+ rats, may be responsible for the increase in growth rate in hepatic neoplasms of female TG+ rats compared with that in livers of male TG+ rats and may contribute to neoplastic progression in the liver of this transgenic model. Published 2004 Wiley‐Liss, Inc.

Hepatocellular carcinomas of the albumin SV40 T‐antigen transgenic rat display fetal‐like re‐expression of Igf2 and deregulation of H19

Previous studies in our laboratory have shown that one of the earliest events during hepatocarcinogenesis in the albumin SV40 T antigen (Alb SV40 T Ag) transgenic rat is the duplication of chromosome 1q3.7‐4.3, a region which contains the imprinted and coordinately regulated genes Igf2 and H19. We have also shown that this duplication is associated with the biallelic expression of the normally monoallelically‐expressed H19. These results, however, are seemingly at odds with studies in the mouse that have shown a conservation of fetal regulatory patterns of these two genes in hepatic neoplasms. We therefore aimed in this study to determine the allelic origin of Igf2 expression in hepatocellular carcinomas of the Alb SV40 T Ag transgenic rat. Sprague–Dawley Alb SV40 T Ag transgenic rats and Brown Norway rats were reciprocally mated and the expression of Igf2 in hepatocellular carcinomas of the resulting F1 transgene‐positive female rats was analyzed by Northern blotting and RT‐PCR. We determined that Igf2 was expressed exclusively from the paternal allele, which prompted the study (by the same methods) of the allelic origin of H19 in the same hepatocellular carcinomas in order to determine if the two genes remained coordinately regulated. Our results demonstrate fetal‐like re‐expression of Igf2 and deregulation of H19 in singular hepatocellular carcinomas of the rat. These results imply that another regulatory mechanism other than the generally accepted ICR/CTCF mechanism may play a role in the control of Igf2 and H19 expression.