Ken Ichi Yamamura

Genomic organization and expression analysis of the mouse qkI locus

Abstract.qkI, encoding a KH domain-containing RNA binding protein, has been isolated as a candidate gene for the mouse neurological mutation quaking. Here, we describe detailed studies on its genomic structure and expression pattern. We isolated approximately 1 Mb of genomic region containing the quaking locus and determined its genomic organization. The qkI locus contains at least 9 exons spanning ∼65 kb of DNA. It gives rise to six distinct transcripts encoding, theoretically, five different protein isoforms. Exons 1 through 4 are shared by all the transcripts, whereas coding exons and two distinct 3′-UTRs downstream to the exon 4 are differentially utilized. One isoform has a truncated KH domain and may act as an antagonist to the others. These findings and identification of a single transcription initiation site suggest that differential expression of each transcript is regulated by alternative splicing. Expression of each alternative transcript and protein product was also examined. Two types of transcripts, 5 kb-A and B, are most abundant in the brain of newborn mice and are gradually downregulated thereafter. In contrast, the other three messages, 6 kb, 7 kb-A and B, increase as myelination proceeds and peak at 2 weeks of age, corresponding to the most active stage of myelination. Although the qkI messages and their products are abundant in brain and heart, a lower level of expression was found in various other tissues tested. Alternative transcripts that share the same 3′-UTR showed very similar expression patterns, suggesting a regulatory role of the 3′-UTRs in qkI gene expression.

Neural and skin cell-specific expression pattern conferred by steel factor regulatory sequence in transgenic mice

We have produced transgenic mice expressing a lacZ reporter gene under the control of a fragment of a Steel factor (SLF). The function of this gene is essential for the development of hematopoietic cells, germ cells, melanocytes and pacemaker cells of the intestine. The expression of the transgene, containing 2 kb DNA 5′ regulatory sequence, was restricted to neural and skin tissues in appropriate spatial and temporal pattern compared with endogenous SLF mRNA expression. This indicates that the regulatory elements necessary for the neural and skin specific expression are present in this 2 kb DNA sequence, although strong position‐dependence of transgene expression was observed. As we could not detect transgene expression in hematopoietic tissues and germ cells after extending 10 kb upstream, elements important for these organs must reside in other regions. Our results indicate that neural crest derived enteric ganglion cells provide SLF to the neighboring pace maker cells expressing c‐kit, the receptor for SLF. Cells expressing the transgene in the intestine are ganglion cells derived from neural crest since homozygosity for the lethal spotting (ls) mutation results in loss of such ganglion cells in transgenic mice. We have also shown that the dermal papillae of the hair follicle expresses the transgene, suggesting its roles to support the c‐kit dependent growth and development of melanocytes in the hair follicle.

The 3′ enhancer region determines the B/T specificity and pro-B/pre-B specificity of immunoglobulin Vκ-Jκ joining

Abstract Using transgenic substrates, we found that the immunoglobulin κ gene 3′ enhancer (E3′) acts as a negative regulator in V κ -J κ joining. Although the E3′ was originally identified as a transcriptional enhancer, it acts in a Buppressive manner for recombinational regulation. Base substitution analysis has shown that the PU. 1-binding site within the E3′ regulates the B/T specificity of V κ -J κ joining. In a substrate with a mutated PU. 1-binding site (GAGGAA to TCTTCG), V κ -J κ joining occurred not only in B cells, but also in T cells. The E3′ region is also responsible for determining the pro-B/pre-B specificity of V κ -J κ joining. When the E3′ region was deleted, κ gene rearrangement actively occurred at the early pro-B stage of B cell development: non-germline (N) nucleotides were common at recombination junctions.

Region-specific expression of murine Hox genes implies the Hox code-mediated patterning of the digestive tract

Hox genes encode transcription factors which are involved in the establishment of regional identities along the anteroposterior (AP) body axis. To elucidate the AP patterning of the digestive tract, we have systematically examined the expression patterns of Hox genes belonging to paralogue groups 6, 7, 8 and 9 by whole‐mount in situ hybridization and by section in situ hybridization analyses.

Cell-cycle-dependent expression of the STK-1 gene encoding a novel murine putative protein kinase

We have cloned a novel putative serine/threonine kinase-encoding gene, designed STK-1, from murine embryonic stem (ES) cell and testis cDNA libraries. The kinase most closely related to STK-1 is Xenopus laevis XLP46 protein kinase which shows 71% amino-acid identity to STK-1 between their kinase domains. Nevertheless, STK-1 is conserved throughout phylogeny with hybridizing sequences being detected in DNA from mammals, amphibians, insects and yeast. STK-1 mRNA is detected in testis, intestine and spleen, tissues that contain a large number of proliferating cells, but not in other tissues. All cell lines tested expressed STK-1 mRNA with levels being dependent upon proliferation rates. In NIH 3T3 cells, STK-1 is expressed in a cell-cycle-dependent fashion. These findings suggest a role for STK-1 in cell growth.

Transgenic mouse expressing a full-length hepatitis C virus cDNA

Hepatitis C virus (HCV), a major causative agent of post transfusion non‐A, non‐B hepatitis (NANBH), can only infect humans and chimpanzees. We produced nine transgenic mouse lines carrying a full‐length HCV cDNA with the human serum amyloid P component (hSAP) promoter that can direct liver‐specific expression. In one of these lines HCV mRNA and HCV core protein were detected in the liver of the transgenic mouse, although the levels of expression were very low. In addition, HCV‐related antibody was detected in the serum.

Comparison of amyloid deposition in two lines of transgenic mouse that model familial amyloidotic polyneuropathy, type I.

We previously produced a transgenic mouse line designated MT-hMet30 by introducing the human mutant transthyretin (TTR) gene carrying the mouse metallothionein promoter, and showed that the presence of human variant TTR is sufficient for amyloid deposition in various tissues of these transgenic mice. However, the expression pattern of human mutant transthyretin gene in the mouse was different from that in man. To analyse pathologic processes, it is essential to establish a transgenic mouse line in which the developmental and tissue- specific expression of the human mutant TTR gene is the same as in man. Thus, we produced two additional transgenic mouse lines carrying the human mutant TTR gene containing either 0.6 kb (0.6- hMet30) or 6.0 kb (6.0-hMet30) of the upstream region. The expression levels of 6.0-hMet30 gene in the liver and serum were the same as in man and about 10 times higher than those of 0.6- hMet30 gene. In both lines amyloid deposition was observed in similar tissues to human patients except for the peripheral and autonomic nervous tissues. The amyloid deposition started earlier and was more extensive in 6.0-hMet30 than 0.6-hMet30 mice, suggesting that the serum levels of human mutant TTR are correlated with the occurrence and degree of amyloid deposition, to some extent. Neither amyloid deposition nor degenerative changes were observed in the peripheral and autonomic nervous systems despite the transgene expression in the choroid plexus of the 6.0-hMet30 mice. In the 6.0-hMet30 mice, amyloid deposition started at 9 months of age, although the serum level of human mutant TTR reached the adult level at 1 month. These results suggest that intrinsic environmental factors other than the mutant gene are involved in the late-onset deposition of amyloid fibrils. Transgenic mice described here should be useful for analysing such factors

A 900 bp genomic region from the mouse dystrophin promoter directs lacZ reporter expression only to the right heart of transgenic mice

In order to study the regulatory mechanism of developmental and tissue‐specific expression of the muscle type dystrophin gene in mice, transgenic mice were generated carrying the 900 bp genomic fragment derived from the muscle type dystrophin promoter region fused to the bacterial lacZgene. Six independent transgenic mouse lines showed specific reporter gene expression in the right heart, but not in skeletal or smooth muscle. The reporter gene expression was first detected in the presumptive right ventricle of the embryos at 8.5 days post coitum, and the expression continued only in the right ventricle throughout the development and at the adult stage. The results indicate that the 900 bp genomic fragment contains the regulatory element required for expression of dystrophin only in the right heart, suggesting that distinct elements are responsible for the expression in the left and right compartments of the heart, and/or in skeletal and smooth muscle cells. Based on these findings, the relationship between defects in muscle type promoter and the diseases caused by abnormal dystrophin expression is discussed.

Molecular and embryological characterization of a new transgene-induced null allele of mouse Brachyury locus

region in wild-type mouse, we found that the pattern was different in the left half of the insert (Fig. 1), suggesting the presence of a junction in the middle of the insert. We then subcloned a BglIIKpnI fragment that might contain the integration junction sequence. DNA sequence analysis of the fragment showed that one end of the insert contained a part of the transgene, while the other end corresponded to the exon 8 of the Brachyury gene. This finding clearly indicated that the #137 mutation represents a new disrupted allele of the Brachyury locus. We hence referred to #137

Comparison of ES cell fate in sandwiched aggregates and co-cultured aggregates during blastocyst formation by monitored GFP expression

Markers and the means to detect them are required to monitor the fate of living cells. However, few suitable markers for living cells were known until a green fluorescent protein (GFP) was discovered. We have established mouse embryonic stem (ES) cell lines that express mutant GFP under the chicken β‐actin (CAG) promoter. Using these cell lines, we were able to follow the migration of ES cells during blastocyst formation both in sandwiching and coculture methods, even if only a single ES cell was used. Furthermore, the contribution of ES cells to the inner cell mass (ICM) was easily estimated at the blastocyst stage. We compared sandwiching with coculture aggregation relative to the contribution of the ES cell in the ICM, and the results indicated that there was no difference in the ratios of chimeric embryos having ICM contributed from cultured ES cells. Furthermore, an aggregated single ES cell was able to contribute three or four cells to the ICM at the blastocyst stage. Thus we conclude that one, instead of two, embryos is enough to make aggregation with ES cells, and a single ES cell attached to an embryo is enough to produce germline chimeras. Moreover, we could clearly observe single cell fate during blastocyst formation. This suggests that our established cell line can be used for monitoring single cell fate in vivo. In addition, we have shown that up to five doses of 30 sec of UV irradiation using GFP filters have no effect on the embryonic development. Mol. Reprod. Dev. 52:376–382, 1999.