SummaryCholesterol deficiency, a new autosomal recessive inherited genetic defect in Holstein cattle, has been recently reported to have an influence on the rearing success of calves. The affected animals show unresponsive diarrhea accompanied by hypocholesterolemia and usually die within the first weeks or months of life. Here, we show that whole genome sequencing combined with the knowledge about the pedigree and inbreeding status of a livestock population facilitates the identification of the causative mutation. We resequenced the entire genomes of an affected calf and a healthy partially inbred male carrying one copy of the critical 2.24‐Mb chromosome 11 segment in its ancestral state and one copy of the same segment with the cholesterol deficiency mutation. We detected a single structural variant, homozygous in the affected case and heterozygous in the non‐affected carrier male. The genetic makeup of this key animal provides extremely strong support for the causality of this mutation. The mutation represents a 1.3kb insertion of a transposable LTR element (ERV2‐1) in the coding sequence of the APOB gene, which leads to truncated transcripts and aberrant splicing. This finding was further supported by RNA sequencing of the liver transcriptome of an affected calf. The encoded apolipoprotein B is an essential apolipoprotein on chylomicrons and low‐density lipoproteins, and therefore, the mutation represents a loss of function mutation similar to autosomal recessive inherited familial hypobetalipoproteinemia‐1 (FHBL1) in humans. Our findings provide a direct gene test to improve selection against this deleterious mutation in Holstein cattle.

The E2F transcription factors have emerged as critical apoptotic effectors. Herein we report that the E2F family member E2F3a can be induced by DNA damage through transcriptional and posttranslational mechanisms. We demonstrate that the posttranslational induction of human E2F3a is dependent on the checkpoint kinases. Moreover, we show that human E2F3a is a substrate for the checkpoint kinases (chk kinases) and that mutation of the chk phosphorylation site eliminates the DNA damage inducibility of the protein. Furthermore, we demonstrate that E2F1 and E2F2 are transcriptionally induced by DNA damage in an E2f3-dependent manner. Finally, using both in vitro and in vivo approaches, we establish that E2f3 is required for DNA damage-induced apoptosis. Thus, our data reveal the novel ability of E2f3 to function as a master regulator of the DNA damage response.The orderly progression through the cell cycle is governed by the coordinated activity of the E2F transcription factors (16). The E2Fs are a family of DNA binding proteins whose activity is regulated through their interaction with the retinoblastoma family (pRb, p107, and p130) (6). The E2Fs regulate a cohort of cell cycle regulatory genes, and they thereby coordinate the transitions through the different phases of the cell division cycle. The 8 E2F proteins (E2F1 to E2F8) are subdivided into two classes based on their transcriptional regulatory activities: the transactivating members (E2F1 to E2F3) and the transrepressing members (E2F4 to E2F8) (6). In general, the transactivating members drive cell cycle progression by inducing the expression of proliferation-associated genes, whereas the transrepressing members impede cell growth by repressing these genes.In addition to regulating cell growth, the E2F transcription factors can promote apoptosis through the activation of deathinducing genes, such as p73, caspases, Apaf1 and Bcl-2 homology region 3 (BH3)-only proteins (9,14,24,25,27,36). Ectopic E2f1 expression can induce both p53-dependent and p53-independent apoptosis (16). Although initial studies had suggested that only E2F1 could induce apoptosis, other studies have demonstrated that E2F2 and E2F3a also possess proapoptotic functions (7,17,31,38). Indeed, the ectopic expression of E2F3a has been shown to promote apoptosis both in vitro and in vivo (5,17,31,38). Interestingly, apoptosis induced by ectopic E2F3 expression can occur in a p53-independent manner (17, 31). However, the absence of E2f1 eliminates the proapoptotic function of E2F3a (17, 31). Thus, one interpretation of these studies is that among the activating E2Fs, only E2F1 has a specific proapoptotic function and that the induction of apoptosis by other E2Fs may be a consequence of deregulated E2F1 activity.Analysis of Rb null embryos has revealed that E2Fs can exhibit proapoptotic activities in a physiological setting (13). Loss of Rb results in extensive apoptosis in developing embryos (15, 21). Genetic deletion of either E2f1 or E2f3 suppresses cell death in the lens of Rb null embryos (39)...

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.