Rat transcobalamin: cloning and regulation of mRNA expression

Abstract

Transcobalamin (TC) has been cloned and used for studying its gene expression in the rat. TC mRNA is distributed widely in adult rat tissues, but at different levels (kidney > liver > lung > yolk sac > intestine > heart > brain > spleen > muscle). TC mRNA levels were 4-fold higher in the jejunum and ileum compared to its levels in the duodenum. During postnatal development, TC mRNA levels in the ileum declined 4-fold from day 4 to day 12, but increased by 5-fold between days 12 and 24. In contrast, TC mRNA levels increased by 2.5-fold in the kidney from day 4 to day 12 and then declined by 2-fold by day 24. Adrenalectomy of adult rats resulted in a 4-fold decline in ileal levels of TC mRNA and a 50% decline in the ileal mucosal formation of the TC-[(57)Co] cobalamin (Cbl) complex following oral administration of [(57)Co]Cbl complexed to gastric intrinsic factor (IF). Cortisone treatment reversed these changes noted in the ileum. In contrast to ileum, kidney TC mRNA levels were not altered significantly in adrenalectomized rats before and after cortisone treatment. Taken together, this study has provided evidence for the regulation of TC gene expression in the rat kidney and intestine during their postnatal development, and cortisone selectively regulates ileal but not kidney TC mRNA levels.

Figure 1. Alignment of amino acid sequence of mature transcobalamin (TC) from the indicated species

The Jellyfish version 1.4 (matrix-Gonnet) program was used for sequence alignment. Consensus residues are indicated in lowercase below each box. The six cysteine residues conserved in identical positions in all four species are indicated by stars above the box, while the two cysteine residues that do not participate in disulphide bond formation are indicated by open stars. The only putatative N-glycosylation site that is not used for N-glycosylation in all the species is indicated by a horizontal bar. The shaded residues represent consensus residues while the residues in bold represent variation in two or more species.

Figure 2. TC mRNA distribution in the rat tissues

Upper panel, mRNA (2 μg) from different adult rat tissues (see lower panel: H, heart; B, brain; S, spleen; L, lung; Li, liver; M, skeletal muscle; K, kidney; I, intestine and Y, yolk sac) was used for Northern blotting. The autoradiograph shown is after a 5 day exposure (CSK, cytoskeletal β-actin mRNA). Lower panel, the ratio of band intensities of TC mRNA and β-actin mRNA are expressed as mean ±s.d. from four separate blots. The ratio of the two transcripts in rat kidney was taken as 100%.

Figure 3. Immuno-crossreactivity of rat TC with human TC antiserum

One millilitre of rat serum bound [⁵⁷Co]Cbl was immunoprecipitated with indicated amounts of human TC antiserum.The radioactivity immunoprecipitated is expressed as percentage of total counts used for immunoprecipitation. The values are mean ±s.d. of four different immunoprecipitation experiments using serum from four separate adult rats. Right panel shows a representative immunoblot generated using rat serum (lane 3), recombinant human TC (lane 2) and antiserum to human TC or preimmune rabbit serum (lane 1).

Figure 4. Distribution of TC mRNA in duodenum, jejunum and ileum of adult rat gut

Upper panel, representative blot of TC mRNA distribution along the different segments of the adult rat intestine (see lower panel: D, duodenum; J, jejunum; I, ileum). Lower panel, the ratio of TC/β-actin mRNA in the three regions; the values shown are mean ±s.e.m. from four separate blots using mRNA isolated from four animals.

Figure 5. Postnatal developmental expression of TC in rat ileum (upper panels) and kidney (lower panels)

The Northern blot obtained using mRNA isolated from rats of indicated ages is shown on the left, and the ratio of TC/β-actin transcripts (•) and the [⁵⁷Co]Cbl binding capacity (▪) of the culture medium of intestinal and renal explants are shown on the right. The values in the right-hand panels are mean ±s.e.m. from five different animals in each age group. Other details are provided in Methods.

Figure 6. Effect of adrenalectomy and cortisone treatment on TC transcript levels in the rat ileum and kidney

Northern blots of TC mRNA in the ileum and kidney of normal rats (lane 1), adrenalectomized rats (lane 2), and adrenalectomized rats treated with cortisone acetate (lane 3) are shown in the upper panels and the ratio of TC/β-actin transcripts in the lower panels. The values are mean ±s.e.m. for six animals from each group.

Figure 7. Plasma apo TC and TC transcript levels in the ileum and kidney during pregnancy in rats

Plasma [⁵⁷Co]Cbl binding capacity (A) and Northern blots of TC transcripts using mRNA from ileum (B) and kidney (C) from non-pregnant females (lane 1), pregnant females (lane 2) and lactating mothers (lane 3). The plasma [⁵⁷Co]Cbl binding capacity shown is mean ±s.e.m. using serum obtained from five animals from each group and the P value with respect to control was >0.05.