• Development and Reproduction
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• v.23 no.4
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• pp.313-322
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• 2019

The epididymal fat pad is a male gonadal adipocyte tissue and is histochemically separated into distal and proximal parts. The development of epididymal fat during postnatal period has not been examined in detail. A previous research showed that expression of adipocyte-associated molecules in the distal epididymal fat of mouse is generally increased as postnatally aged. In the present study, expressional patterns of same adipocyte-associated molecules in the mouse proximal epididymal fat at 2, 5, 8, and 12 months of age were studied by quantitative real-time PCR analysis and were compared with those in the distal epididymal fat. The expressional levels of peroxisome proliferator-activated receptor gamma (Pparg), lipoprotein lipase (Lpl), and fatty acid synthase (Fasn) at 5 months of age were significantly lower than those at 2 months of age, while transcript level of leptin (Lep) at 5 months was higher than that at 2 months of age. The transcript levels of all molecules at 8 months of age were significantly increased, compared with those at 2 and 5 months of age. At 12 months of age, expression of delta like non-canonical Notch ligand 1 (Dlk1) was further significantly increased, while there was no change on the transcript level of Pparg and significant decreases of Fabp4, Retn, Lpl, Lep, Fasn, and adiponectin (Adipoq) transcript levels. The current findings show that expressional patterns of molecules associated with adipocyte in the proximal epididymal fat is somewhat different with those of the distal epididymal fat, suggesting the existence of regional variance in the epididymal fat.

• BMB Reports
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• v.46 no.3
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• pp.163-168
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• 2013

The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outward-facing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription.

• Development and Reproduction
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• v.27 no.4
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• pp.175-183
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• 2023

The epididymal fat is a type of gonadal adipose tissue, which is localized closely to the testis. Even though it has been suggested that the epididymal fat is necessary for maintenance of spermatogenesis in the testis, the influence of epididymal fat on expression of testicular steroidogenic enzymes has not been examined. In the present research, expressional changes of steroidogenic enzymes in the mouse testis after 2 weeks of the surgical partial lipectomy of epididymal fat at different postnatal ages were determined by real-time polymerase chain reaction analysis. The transcript levels of all molecules at 2 months of postnatal age were significantly increased by the lipectomy of epididymal fat. However, the lipectomy at 5 months of postnatal age resulted in decreases of expression levels of all molecules examined in the testis. Except a reduced transcript level of hydroxysteroid 17-beta dehydrogenase 3, there were no significant changes of expression levels of other steroidogenic enzymes by the lipectomy at 8 months of postnatal age. At 12 months of postnatal age, the lipectomy caused a significant increase of transcript level of steroidogenic acute regulatory protein and a significant decrease of transcript level of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1, without any expressional change of cytochrome P450 side chain cleavage, hydroxysteroid 17-beta dehydrogenase 3, and hydroxysteroid 17-beta dehydrogenase 3 in the testis. These findings suggest that the substances derived from epididymal fat could differentially influence on expression of steroidogenic enzymes in the testis during postnatal period.

• Animal cells and systems
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• v.5 no.1
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• pp.71-75
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• 2001

Spin is an abundant maternal transcript comprising up to 0.2% of the total mRNA stock in mouse oocyte, whose protein product is associated with the meiotic spindle. We have identified a new isoform of Spin transcript containing a distinct 5'-untranslated region and the N-terminus of encoded protein. Northern blot and RT-PCR analysis showed that the new isoform is expressed in embryos and most of adult tissues, while the previously identified transcript is expressed solely in mouse oocyte. We thus designated these two Spin isoforms as somatic type and oocyte type, respectively. To investigate the underlying mechanism for the differential expression, genomic structure of Spin was examined. Spin exists as multiple copies in the genome, some of which appears to be pseudogenes, and characterization of Spin genomic clones indicates that oocyte- and somatic-isoforms were generated by alternative splicing. The complex organization of Spin genomic locus and its multifaceted control of expression provide a good model to study the molecular mechanisms of elaborate genome usage in mammals.

• Genomics & Informatics
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• v.4 no.1
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• pp.33-39
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• 2006

Alternative splicing (AS) is an important mechanism of producing transcriptome diversity and microarray techniques are being used increasingly to monitor the splice variants. There exist three types of microarrays interrogating AS events-junction, exon, and tiling arrays. Junction probes have the advantage of monitoring the splice site directly. Johnson et al., performed a genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays (Science 302:2141-2144, 2003), which monitored splicing at every known exon-exon junctions for more than 10,000 multi-exon human genes in 52 tissues and cell lines. Here, we describe an algorithm to deduce the relative concentration of isoforms from the junction array data. Non-negative Matrix Factorization (NMF) is applied to obtain the transcript structure inferred from the expression data. Then we choose the transcript models consistent with the ECgene model of alternative splicing which is based on mRNA and EST alignment. The probe-transcript matrix is constructed using the NMF-consistent ECgene transcripts, and the isoform abundance is deduced from the non-negative least squares (NNLS) fitting of experimental data. Our method can be easily extended to other types of microarrays with exon or junction probes.

• BMB Reports
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• v.29 no.2
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• pp.127-132
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• 1996

Germinal centers (GCs) are formed in peripheral lymphoid tissues in response to protein antigens. In order to see if immunoglobulin isotype switching takes place in GC B-cells, we isolated GC B-cells (PNA positive cells) from mouse popliteal lymph nodes by a flow cytometer after the staining of lymph node cells with PNA-FITC and anti-B220-PE, and determined the expression of ${\gamma}1$ germline transcript and ${\gamma}1$ mRNA by RT-PCR. ${\gamma}1$ germline transcript and ${\gamma}1$ mRNA were amplified specifically in cDNAs from hybridoma expressing IgG1 or splenocytes stimulated LPS plus IL-4. Germinal center B-cells formed in popliteal lymph nodes of mice immunized with chicken ovalbumin were isolated 7 days after immunization. We sorted GC B-cells five times. Immunoglobulin ${\gamma}1$ germline transcripts were expressed in germinal center B-cells in three out of five sorts whereas two out of five sorts did not express ${\gamma}1$ germline transcripts in GC B-cells. The contents of GC B-cells ranged from 5 to 7% of total lymph node cells in most flow cytometric analyses but those of two sorted cells which did not express ${\gamma}1$ germline transcripts were out of normal range. These results imply that isotype switching of immunoglobulins may take place in GCs.

• Journal of Animal Science and Technology
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• v.54 no.6
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• pp.419-425
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• 2012

The coordinated action of the detrusor muscle cells in the urinary bladder is governed by cell-cell communication through gap junction, consisted of connexin (Cx) molecules. Even though a number of researches have been mostly focused on expressional changes of a few Cx isoforms in clinically dysfunctional condition of the bladder, less attention has been paid for investigation of Cx isoforms present in the bladder. Using real-time PCR analysis, the present study examined Cx isoforms expressing in the male rat bladder during postnatal period. Also, expressional patterns of Cx isoforms were evaluated in the bladder at different postnatal ages. Of a total of 13 Cx isoforms tested in the present study, we were able to detect mRNAs of 6 Cx isoforms in the rat urinary bladder, including Cxs 31, 31.1, 32, 37, 40, and 45. The transcript levels of Cxs 31, 31.1, 37, 40, and 45 were gradually increased from 1 week of age until 25 days of age, followed by transient decreases at 45 days of age. However, abundance of Cx32 transcript was drastically increased at 15 days of age, followed by a sharp drop at 45 days of age. These results indicate that differential expression of Cx isoforms in the bladder during postnatal development would be necessary for maintaining proper function of the bladder. A question remains to be answered if significant decreases of transcript levels of some Cx isoforms at the elderly are associated with age-dependent dysfunction of the bladder.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.5
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• pp.1937-1941
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• 2014

Aims: To evaluate anti-cancer activity of Asparagus racemosus (AR) leaf extract on UOK146, a renal cell carcinoma cell line, and explore its mechanism of action. Materials and Methods: Dried AR leaves were extracted with chloroform and dissolved in DMSO. This extract was applied to UOK146 and cell death was estimated by MTT assay. In addition PRCC-TFE3 fusion transcripts were detected by real time PCR. Results: Extract was found to be cytotoxic with an $IC_{50}$ of 0.9 mg/ml as estimated by dose response curve. Antitumor activity of the permissible doses of the extract was assessed by the down regulation of PRCC-TFE3 fusion transcript (38%) responsible for oncogenicity of the UOK146 cell line. No increment in the BAX, a proapoptotic marker level was observed. Conclusions: Evidence of antiproliferative effect, PRCC-TFE3 fusion transcript inhibition and static BAX level clearly indicate that AR extract provides or elicits an apoptosis independent anticancer effect on RCC cells by some specific mechanism of regulation.

• Proceedings of the Korea Information Processing Society Conference
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• 2012.11a
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• pp.1326-1329
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• 2012

전사체(transcript)는 유전자로부터 전사된 DNA 시퀀스 코드를 말한다. 전사체(transcript)의 발현된 형태에 따라 생성되는 단백질의 형태 역시 달라지므로 전사체 모델의 형태는 중요한 의미를 가지며 특정 위치의 전사체가 정상과 다르게 모델이 변할 경우 심각한 경우에는 유전자 질병에 노출될 수 있다. 현재 실험체에 대한 전사체 모형은 SpliceGrapher, Cufflinks와 같은 상용화된 도구들을 사용하여 얻을 수 있다. 하지만 이런 도구 간의 결과 값 및 어노테이션 정보와 결과 값 간의 유사도 비교를 위한 방법론은 현재 알려진 바 없다. 대신 전사체 비교를 위해 모형 간의 차이를 눈으로 하나씩 비교하거나 전사체 위치를 이용한 산수 값을 이용한다. 본 논문에서는 전사체 모형 간의 유사도를 비교하기 위한 방법론을 제시하고 Homo sapiens grch37 어노테이션 파일과 SRR387514 실험 데이터 간의 유사도를 제시한 방법론을 이용하여 측정한 결과 값을 분석하였다.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.829-835
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• 2000

The ptsH and ptsI genes of Lactococus lactis subsp. lactis ATCC 7962 (L. lactis 7962), encoding the general proteins of phosphotransferase system (PTS) components, HPr and enzyme I, respectively, were cloned and characterized. A 1.3 kb PCR product was obtained using a primer set that was hybridized to the internal region of the L. lactis 7962 pts HI genes and then subcloned into a low-copy number vector, pACYC184. The 5' upstream and 3' downstream region from the 1.3 kb fragment were subsequently clone using the chromosome walking method. The complete ptsHI operon was constructed and the nucleotide sequences determined. Two ORFs corresponding to HPr (88 amino acids) and enzyme I (575 amino acids) were located. The ptsHI genes of L. lactis 7962 showed a very high homology (84-90%) with those genes from other Gram-positive bacteria. A primer extension analysis showed that the transcription started at either one of two adjacent bases upstream of the start codon. Using a Northern analysis, two transcripts were detected; the first, a 0.3 kb transcript corresponding to ptsH and the second, a 2 kb transcript corresponding to ptsH and ptsI. The transcription level of ptsH was higher than that of ptsI. The concentration of the ptsH transcript in cells grown on glucose was similar to that in cells grown on lactose, yet higher than that in cells grown on galactose. The ptsI transcript was scarcely detected in cell grown on lactose or galactose. The ptsI transcript was scarcely detected in cells grown on lactose or galactose. The results of a sequence analysis and Northern blot confirmed that the ptsH and ptsI genes of L. lactis 7962 were arranged in an operon like other known ptsHI genes and the expression of the ptsHI genes was regulated at the transcriptional level in response to the carbon source.